Prophylactic and therapeutic immunization against protozoan infection and disease

ABSTRACT

Polypeptide and polynucleotide vaccines effective to treat or prevent infection of a mammal, such as a dog, a cat, or a human, by a protozoan. Methods of treatment and prevention are also provided, including therapeutic administration of the vaccine to an infected mammal to prevent progression of infection to a chronic debilitating disease state. Preferred embodiments of the polynucleotide vaccine contain nucleotide coding regions that encode polypeptides that are surface-associated or secreted by  T. cruzi . Optionally the efficacy of the polynucleotide vaccine is increased by inclusion of a nucleotide coding region encoding a cytokine. Preferred embodiments of the polypeptide vaccine include immunogenic peptides that contain membrane transducing sequences that allow the polypeptides to translocate across a mammalian cell membrane.

This application is a continuation of U.S. Ser. No. 11/893,951, filed onAug. 17, 2007, which is a divisional patent application of U.S. Ser. No.11/015,578, filed on Dec. 17, 2004, now U.S. Pat. No. 7,309,784, whichis a divisional application of U.S. patent application Ser. No.09/518,156, filed Mar. 2, 2000, now U.S. Pat. No. 6,875,584, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/122,532,filed Mar. 2, 1999, each of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grant numbers RO1AI22070 and AI33106 from the National Institutes of Health. The U.S.government has certain rights in this invention.

BACKGROUND

The etiologic agent of Chagas' disease is an obligate intracellularprotozoan parasite, Trypanosoma cruzi. In mammalian hosts T. cruzicycles between a trypomastigote stage which circulates in the blood andthe amastigote stage which replicates in the cytoplasm of infected hostcells (primarily muscle). Chagas' disease is prevalent in almost allLatin American countries including Mexico and Central America, whereapproximately 18 million people are infected with T. cruzi and roughly50,000 children and adults die of chronic Chagas' disease every year dueto lack of effective treatments. More than 90 million are at risk ofinfection in endemic areas. Additionally, 2-5% of fetus carried byinfected mothers in endemic areas are either aborted or born withcongenital Chagas' disease. Loss of revenue in terms of productivitylost due to sickness and medical costs have an overwhelming effect oneconomic growth of these countries. In the U.S., 50-100 thousandserologically positive persons progressing to the chronic phase ofChagas' disease are present, and the number of infected immigrants indeveloped countries is increasing. Therefore, the risk of transmissionof T. cruzi to non-infected individuals through blood transfusion andorgan transplants from the infected immigrant donors exists.

Attempts to control the vector have been made in an effort to control orprevent T. cruzi infection. Government funded programs for the reduviidvector control and blood bank screening in the developing South Americancountries have been effective in reducing the transmission of T. cruzi.However, the operational costs to maintain such control programs,behavioral differences among vector species, the existence of animalreservoirs, and the persistence of parasites in chronically infectedpatients prevent these control measures alone from completelycontrolling T. cruzi infection.

Chemotherapeutic treatments, a potential means by which parasite load inthe acute or chronic phase of the disease development and thereby theseverity of disease can be reduced, have been partially successful incontrolling T. cruzi infection and Chagas' disease. However, the hightoxicity of drugs and poor efficacy of available therapeutics havecombined to limit the utility of chemotherapy for treatment of bothacute and chronic patients. Further, drug therapy reduces the severityof disease in chronically infected individuals, but cannot reverse thedamage already done by parasites.

Vaccines for prevention or treatment of T. cruzi infection arepractically non-existent. Traditional vaccines constituted ofheat-inactivated parasites, or subcellular fractions of T. cruzi providea degree of protection from T. cruzi infection (M. Basombrio, Exp.Parasitol. 71:1-8 (1990); A. Ruiz et al., Mol. Biochem. Parasitol.39:117-125. (1990)). However, these vaccines failed to elicit theprotective level of immunity, probably due to loss of important epitopesduring inactivation and/or the failure of the antigens to enter the MHCclass I pathway of antigen processing and presentation and elicit cellmediated immune responses (J. Monaco, Immunol. Today 13:173-179 (1992)).Live attenuated vaccines are capable of entering the MHC class Ipathway, and might elicit protective immune responses. However, thedanger of reversion of attenuated parasites to virulent strains ifattenuation is not complete renders these vaccines impractical. A DNAvaccine containing the gene encoding a trans-sialiadase has been shownto provide prophylactic protection against T. cruzi infection in mice(F. Costa et al., Vaccine 16:768-774 (1998)), but has not been shown toprevent or reverse disease or to stimulate a CD8⁺ T cell response in theanimal. In another report, specific cellular and humoral immune responsein BALB/c mice immunized with an expression genomic library of T. cruziwas observed (E. Alberti et al., Vaccine 16:608-612 (1998)).

Most vaccine research has centered on attempts to develop prophylacticprotein vaccines against T. cruzi infection, and has met with littlesuccess. The development of subunit vaccines composed of definedantigens which are capable of inducing strong humoral and type 1 T cellresponses and reducing the parasite burden has been hindered by the lackof knowledge of the biology of the three developmental stages of T.cruzi, the lack of sufficient sequence information on genes expressed inthe infective and intracellular stages, and the prevailing scientificview that chronic disease is not associated with persistent parasiticinfection but is the result of a parasite-induced autoimmune response.The presence of polyclonal activation of B and T cells during the acutephase of infection, the difficulty in demonstrating the existence of T.cruzi in the hearts of hosts with severe cardiac inflammation, and thepresence of antigens that are shared or cross-reactive between heart andparasites have been used to promote the idea that anti-heart autoimmunelymphocyte cytotoxicity or humoral immune reactions are responsible forthe development of Chagas' disease. A corollary to this view is thatvaccination against T. cruzi infection or boosting the immune responseof infected individuals will exacerbate the disease. On the other hand,immunohistochemical detection of the T. cruzi antigens or detection ofT. cruzi DNA by sensitive in situ PCR or reverse transcriptase (RT)-PCRtechniques in chronic chagasic cardiopathy in murine models (Y. Gomes,Appl. Biochem. Biotechnol. 66:107-119 (1997); E. Jones et al., Am. J.Trop. Med. Hyg. 48:348-57 (1993); M. Reis et al., Clin. Immunol.Immunopathol. 83(2):165-172 (1997)) as well as humans (J. Lane et al.,Am. J. Trop. Med. Hyg. 56:588-595 (1997)) has been reported. Also, adirect correlation between myocardial inflammatory infiltrates and thepresence of parasites and development of chronic heart failure in amurine model using heart transplantation (R. Tarleton et al., Proc.Natl. Acad. Sci. USA. 94:3932-3937 (1997)), and in chagasic patientsusing endomyocardial biopsies (M. Higuchi et al., Clin. Cardiol.10:665-670 (1987)) has been demonstrated. See R. Tarleton et al.,Parasitology Today 15:94 (1999) for a review.

SUMMARY OF THE INVENTION

The present invention is directed to prophylactic and therapeuticimmunization of mammals against protozoan infection and disease. Medicaluses in humans to prevent or treat protozoan infection, and veterinaryuses in other animals to prevent or treat protozoan infection or tocontrol transmission of infection are examples of contemplatedapplications.

In one aspect, the invention provides a vaccine that is effective totreat or prevent infection of a mammal by a protozoan. Examples ofprotozoans against which a vaccine of the invention is effective includeTrypanosoma, Leishmania, Toxoplasma, Eimeria, Neospora, Cyclospora andCryptosporidia. In a particularly preferred embodiment, the vaccine iseffective against T. cruzi infection and/or disease caused by T. cruzi.The vaccine can be a polypeptide vaccine or a polynucleotide vaccine,and can include one or more immunogenic components. A polynucleotidevaccine contains one or more polynucleotides containing a nucleotidecoding region that encodes an immunogenic polypeptide derived from theprotozoan. Analogously, a polypeptide vaccine contains one or moreimmunogenic polypeptides derived from the protozoan.

The immunogenic polypeptide included in the vaccine or encoded by anucleotide coding region included in the vaccine is preferably asurface-associated polypeptide, such as a GPI-anchored polypeptide, or asecreted polypeptide. In embodiments of the vaccine targeted to T.cruzi, the immunogenic polypeptide is preferably one that is expressedin a T. cruzi amastigote.

The vaccine of the invention preferably stimulates an antibody responseor a cell-mediated immune response, or both, in the mammal to which itis administered. More preferably the vaccine stimulate a Th1-biased CD4⁺T cell response or a CD8⁺ T cell response; most preferably, especiallyin the case of a single component vaccine, the vaccine stimulates anantibody response, a Th1-biased CD4⁺ T cell response and a CD8⁺ T cellresponse. A particularly preferred embodiment of the polynucleotidevaccine of the invention includes a nucleotide coding region encoding acytokine, to provide additional stimulation to the immune system of themammal. A particularly preferred embodiment of the polypeptide vaccineof the invention includes an immunogenic polypeptide that contains amembrane translocating sequence, to facilitate introduction of thepolypeptide into the mammalian cell and subsequent stimulation of thecell-mediated immune response.

Pharmaceutical compositions containing immunogenic polypeptides orpolynucleotides encoding immunogenic polypepdtides together with apharmaceutical carrier are also provided.

In another aspect, the invention provides a recombinant method of makinga vaccine that is effective to treat or prevent infection of a mammal bya protozoan. For example, a multicomponent polynucleotide vaccine ismade by inserting two or more nucleotide coding regions encoding animmunogenic polypeptide derived from the protozoan into two or morepolynucleotide vectors, then combining the polynucleotide vectors toyield a polynucleotide vaccine. In another example, a multicomponentpolypeptide vaccine is made using two or more expression vectors thatcontain a nucleotide coding region that encodes a membrane transducingsequence, into which nucleotide coding regions encoding an immunogenicpolypeptide derived from the protozoan have been inserted in frame. Thisyields a construct encoding an immunogenic fusion protein that containsmembrane transducing sequence linked to the immunogenic polypeptide.Suitable host cells are transformed with the resulting expressionvectors, and expression of the immunogenic fusion proteins is initiated.The fusion proteins are purified, optionally destabilized with urea,then combined to yield a polypeptide vaccine.

In another aspect, the invention provides methods for treating orpreventing infection of a mammal by a protozoan. A vaccine of theinvention can, for example, be administered therapeutically to a mammalharboring a persistent protozoan infection. In one embodiment of thetherapeutic administration of the vaccine, administration of the vaccineis effective to eliminate the parasite from the mammal; in anotherembodiment, administration of the vaccine is effective to prevent ordelay chronic debilitating disease in the mammal. Alternatively, avaccine of the invention can be administered prophylactically to amammal in advance of infection by the protozoan. In one embodiment ofthe prophylactic administration of the vaccine, administration of thevaccine is effective to prevent subsequent infection of the mammal bythe protozoan. In another embodiment, administration of the vaccine iseffective to prevent the development of chronic debilitating disease themammal after subsequent infection by the protozoan. In yet anotherembodiment, administration of the vaccine effective to prevent the deathof the mammal after subsequent infection by the protozoan.

The method of treating or preventing protozoan infection of a mammalalso envisions administering both polynucleotide and polypeptidevaccines prophylactically or therapeutically to a mammal in a protocolthat includes multiple administrations of the vaccine. For example, themammal can be first immunized with a polynucleotide vaccine of theinvention, then boosted at a later time with a polypeptide vaccine.Different types of vaccines (i.e., polynucleotide or polypeptidevaccines), or vaccines of a single type containing different components(e.g., plasmid DNA, viral DNA, vaccines including or encoding differentimmunogenic polypeptides, with or without cytokines or adjuvants) can beadministered in any order desired. An example of a serial protocolinvolves first administering to a mammal a plasmid DNA vaccine, thenlater administering a polypeptide vaccine or viral vector vaccine.Another example involves first administering to the mammal a viralvector vaccine, followed by administering a polypeptide vaccine.

In another aspect, the invention includes a method for identifyingimmunogenic protozoan polypeptides from a protozoan genomic library, foruse in a polynucleotide vaccine. In one embodiment, the method utilizesexpression library immunization (ELI) in mice to identify protozoanpolypeptides that elicit an immune response in a mammal effective toprevent the death of the mammal or to arrest or delay the progression ofdisease in the mammal associated with infection of the mammal by theprotozoan. Preferably, the method is used to identify immunogenicpolypeptides derived from T. cruzi, and BALB/c or B6 mice are immunized.In another embodiment, the method involves

(a) preparing a DNA microarray comprising open reading frames of T.cruzi genes;

(b) preparing a first probe comprising Cy3-labeledtrypomastigote-derived T. cruzi cDNA;

(c) preparing a second probe comprising Cy5-labeled amastigote-derivedcDNA;

(d) cohybridizing the first and second probes to the microarray toidentify at least one gene whose expression is upregulated in T. cruziduring the intracellular amastigote stage of the infectious cycle, whichgene encodes a candidate immunogenic T. cruzi polypeptide; and

(e) immunizing mice with the gene to determine whether the gene encodesa T. cruzi polypeptide that elicits an immune response in a mammaleffective to prevent the death of the mammal or to arrest or delay theprogression of disease in the mammal associated with infection of themammal by T. cruzi.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows T. cruzi-specific serum antibody response in TSA-1 DNAvaccinated BALB/c and B6 mice. The presence of parasite-specificantibodies was assessed by ELISA using a 1:100 dilution of sera pooledfrom individual tail blood samples (4 to 5 mice per group) and collected3 and 2 weeks after first (1) and second (2) vaccination. Negative andpositive controls were sera from normal mice (NMS) and from mice acutelyinfected with T. cruzi (TcIS).

FIG. 2 shows induction of long-lasting TSA-1₅₁₅₋₅₂₂-specific, CD8⁺ Tcell-dependent, MHC class I-restricted CTL in TSA-1 DNA-immunized B6mice; (A) Immune splenocytes obtained 2 weeks after the secondvaccination; (B) Immune splenocytes from DNA vaccinated or T.cruzi-infected mice were obtained 7 and 6 months after the secondvaccination or parasite challenge, respectively; E:T represents theratio of effector cell to target cell.

FIG. 3 shows induction of parasite-specific, MHC class I-restricted andCD8⁺ T cell-dependent CTL response in TSA-1 DNA-immunized BALB/c mice;(A) unstimulated effectors and (B) infected J774-stimulated effectors.

FIG. 4 shows (A) parasitemia and (B) mortality in TSA-1 plasmid DNAvaccinated B6 mice. Values represent mean±SEM in surviving mice.

FIG. 5 shows (A) parasitemia and (B) mortality in TSA-1 plasmid DNAvaccinated BALB/c mice.

FIG. 6 is a schematic of plasmid pCMVI.UBF3/2 engineered to containTSA-1, ASP-1 or ASP-2.

FIG. 7 shows that humoral immunity induced by intramuscular immunizationof B6 mice with T. cruzi antigen-encoding plasmids is enhanced bycytokine adjuvants; (A) T. cruzi specific antibody levels; (B) Antibodysub-types in mice immunized with genetic vaccines.

FIG. 8 shows induction of cellular immunity by intra-muscularimmunization of B6 mice with T. cruzi antigen-encoding plasmids; (A)Percent specific lysis as an indicator of CTL activity in mice immunizedwith T. cruzi antigen-encoding vectors; (B) Induction of T. cruzispecific CTL activity by genetic vaccines is augmented by co-injectionof cytokine encoding vectors; (C) Serum level of IFN-γ in DNA immunizedmice.

FIG. 9 shows (A, C) parasitemia and (B, D) mortality in mice immunizedwith DNA vaccine containing plasmid CMVI.UBF3/2 encoding ASP-1, ASP-2 orTSA-1 (C, D) with or (A, B) without cytokine adjuvants.

FIG. 10 shows that elicitation of protective immune responses bymulti-component nucleic acid vaccine can be augmented by cytokines. Twoweeks after the second immunization, measurements of (A) serum antibodylevels (using ELISA) and (B-E) CTL activity were made. For quantitationof CTL activity, splenocytes from immunized mice were stimulated invitro with antigen-specific peptide (B) PA8, (C) PA14, (D) pep77.2 or(E) a mixture of PA8⁺PA14+pep77.2 (E). Effectors generated from thesesplenocytes were then tested in a 5 hour ⁵¹Cr release assay againstRMA-S target cells sensitized with either the homologous peptide (PA14,PA8 or pep77.2, open symbols) or pulsed with non-specific peptide(OVA₂₅₇₋₂₆₄, filled symbols).

FIG. 11 shows (A) parasitemia and (B) mortality in mice immunized withmulti-component nucleic acid vaccine and infected with T. cruzi.

FIG. 12 shows hematoxylin and eosin stained tissue sections fromskeletal muscles of DNA immunized mice during acute phase of T. cruziinfection. C57BL/6 mice were immunized with (A) vector DNA, (B) cytokineplasmids, (C) antigen-encoding vectors, and (D) a mixture of ASP-1,ASP-2, TSA-1 expression constructs+cytokine expressing plasmid twice at6-week intervals. Tissue sections for histological analysis wereobtained 45 dpi. Parasite infected cells are indicated by arrows.Original magnification 200×.

FIG. 13 shows control of tissue inflammation and parasite burden byprophylactic DNA immunization. Histological analysis of skeletal musclesof mice immunized with (A) vector alone, (B) cytokine-expressingplasmids, (C) ASP-1, ASP-2 and TSA-1-encoding plasmids, or (D) a mixtureof antigen-encoding constructs plus cytokine adjuvants.

FIG. 14 shows the effect of depletion of T cell population on protectiveefficacy of DNA vaccines against T. cruzi infection; (A) parasitemia;(B) mortality.

FIG. 15 shows control of tissue inflammation and parasite burden bytherapeutic DNA immunization. Histological analysis of skeletal musclesof mice immunized with (A) vector alone, (B) cytokine-expressingplasmids, (C) ASP-1, ASP-2 and TSA-1-encoding plasmids, or (D) a mixtureof antigen-encoding constructs plus cytokine adjuvants.

FIG. 16 shows mortality for B6 mice vaccinated with antigen (ASP-1,ASP-2 and TSA-1) encoding vectors (B) with or (A) without cytokines(IL-12 and GM-CSF) expression constructs as a function of dayspost-infection (dpi).

FIG. 17 shows (A) T. cruzi-specific serum antibody levels in C3H micevaccinated with antigen (ASP-1, ASP-2 and TSA-1) encoding vectors withand without cytokines (IL-12 and GM-CSF), together with (B) bloodparasite levels and (C) mortality as a function of days post-infection.

FIG. 18 is a schematic of plasmid pTAT/pTAT-HA used produce polypeptidescapable of translocating across mammalian plasma membranes.

FIG. 19 shows the coding region of T. cruzi Lyt protein (porin).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Over 100 natural mammalian hosts are known for T. cruzi, and T. cruzican be transmitted to a human from another animal host. Any mammalianhost can be immunized in accordance with the invention. Preferredvaccine recipients include humans, domestic animals such as dogs andcats, rodents and wildlife animals. Preferably, the mammal that isimmunized is a dog, a cat, or a human.

Humoral (Antibody-Mediated) and Cell-Mediated Immunity to T. cruzi

In the mammalian host, T. cruzi cycles between a dividing intracellularstage (the amastigote) and a non-replicative extracellulartrypomastigote than which circulates in the blood. The presence of twodevelopmental stages of T. cruzi in mammalian hosts provides twoanatomically and (to some degree) antigenically distinct targets ofimmune detection—the trypomastigotes in the bloodstream and theamastigotes in the cytoplasm of infected cells. The intracellularlocation of amastigotes of T. cruzi has long been considered a “hidingplace” for the parasite wherein it is not susceptible to immunerecognition and control. Thus, studies of immunity to T. cruzi havelargely centered around the recognition and elimination of theextracellular stage.

The present invention challenges the conventional approach ofvaccinating against trypomastigotes by providing, in a preferredembodiment, compositions and methods by which immune responses toamastigotes can be potentiated by therapeutic or prophylacticpolynucleotide or polypeptide vaccines. To this end, the approach of thepresent invention involves stimulating or promoting immune recognitionof the infected host cell and the antigens involved in this recognition.Convention is further challenged by the scientific basis of the presentinvention: that disease development following initial T. cruzi infectionis not an autoimmune response but instead is dependent upon thepersistent presence of parasites in tissues. Previous studies based onthe autoimmune theory of disease development predicted that geneticimmunization against T. cruzi would exacerbate disease development,however it is shown in Example II that genetic immunization of miceprior to exposure to T. cruzi reduces disease development.

The antibody response to T. cruzi trypomastigotes has been well-studiedand numerous specific targets of this response described. However,attempts to vaccinate experimental hosts using these target molecules inprotocols that elicit primarily antibody responses (accompanied bylittle to no cellular immune response) have not been very fruitful.Without intending to be bound by theory, it is proposed that althoughantibodies may be necessary for control of T. cruzi infection, theirrole is likely secondary in importance to cell-mediated immuneresponses. For example, mice lacking the ability to make antibodies byvirtue of the muMT targeted deletion live significantly longer than domice lacking either CD4⁺ or CD8⁺ T cells but do eventually die, evenwhen infected with very low numbers of parasites. It is of coursepossible that vaccine-induced antibody responses fail to provide goodprotection because the wrong target molecules are used for theimmunization or because non-protective isotypes of antibodies areelicited by the immunization. However, we believe it is more likely thatprotective immunity cannot be achieved by induction of parasite-specificantibodies alone; instead, this antibody response is preferablyaccompanied by a potent cell-mediated immune response.

Induction of T helper cell responses skewed toward the production oftype 1 cytokines such as IL-2 and IFN-γ are also of substantialimportance in immunity to T. cruzi. The Th1 set of cytokines enhancemacrophage activation for killing of T. cruzi via a nitric oxide (NO)dependent mechanism (e.g., J. Silva et al., Infect. Immure. 63:4862-4867(1997)) and are also likely to be important in the induction ofprotective antibody responses and as helper factors in the cytotoxicT-lymphocyte (CTL) response. Infections in mice depleted of Th1cytokines and in gene-knockout mice provide solid evidence that Th1responses associate with protection from T. cruzi and Th2 responsesassociate with susceptibility. In our hands, mice lacking the ability toproduce type 1 cytokines by virtue of the absence of the STAT4 geneproduct are extremely susceptible to T. cruzi infection while STAT6knockout mice which fail to make type 2 cytokines are more resistant toinfection than are wild-type mice (R. Tarleton et al., ParasitologyToday 15:94-99 (1999)).

It is demonstrated herein that a third effector mechanism is importantto immune control of T. cruzi, i.e., the class I MHC-restricted CD8⁺ Tcell response. Cytoplasmic pathogens are recognized by the immune systemvia class I MHC-presented peptides. During the processes ofdifferentiation, replication and metabolism, T. cruzi releases a varietyof proteins into the host cell. All cytoplasmic proteins are susceptibleto degradation by the proteasome and the resulting peptides can betransported into the endoplasmic reticulum via transporters associatedwith antigen processing (TAPs). In vertebrate cells, these peptides havethe potential to associate with newly synthesized class I majorhistocompatibility complex (MHC) molecules and to be displayed as classI MHC-peptide complexes on the cell surface. This process allowsessentially all vertebrate cells to sample the contents of the cytoplasmand to display to the immune system portions of the proteins beingexpressed within. In the case of normal cellular proteins, the peptidesdisplayed in association with class I MHC are presumably ignored by theimmune system. However when peptides from mutated self proteins or fromproteins encoded by invading viruses, bacteria or protozoans aredisplayed, CD8⁺ T cells capable of recognizing the foreign peptide-selfMHC complex can initiate the responses capable of killing the infectedcells or controlling the growth of the pathogen within. TSA-1, ASP-1 andASP-2, GPI-anchored proteins expressed in T. cruzi primarily during theamastigote stage are examples of targets of the CTL response in T. cruziinfected mice (Example II). Humans with chronic T. cruzi infectionsrecognize these same target antigens (B. Wizel et al., J. Clin. Invest.102:1062-71 (1998)). It should be understood that although most CD8⁺ Tcells are cytotoxic T-cell lymphocytes (CTLs) some can, alternatively orin addition, produce cytokines, and this cytokine-producing activity mayalso be an important result of the stimulation of a CD8⁺ T cell immuneresponse.

Without intending to be bound by theory, it is believed that proteinsanchored by glycosylphosphatidylinositol (GPI) in T. cruzi may stimulateall three of these distinct immune responses, thereby generating abroader protection against T. cruzi than was previously possible. Thefailure of previous attempts at vaccination in T. cruzi is likely due tothe fact that investigators have largely focused on induction of onlyone or two of these responses, principally antibody production and, to alesser extent, CD4⁺ T cell responses. The majority of surface proteinsin trypomastigotes and amastigotes of T. cruzi are GPI-anchored and manyof these surface proteins both elicit and are bound by antibodies. Inaddition, the GPI anchoring mechanism in T. cruzi appears to be verysloppy with a significant portion of proteins targeted for GPI additionbeing secreted without the addition of a GPI anchor (N. Garg et al., J.Biol. Chem. 272:12482-12491 (1997)). In the case of amastigotes, thesesecreted proteins lacking GPIs enter the host cell cytoplasm, arepresented by class I MHC molecules and elicit the production of CTLresponses. On extracellular amastigotes and trypomastigotes, these sameproteins in a surface-anchored fowl sensitize parasites to detection byantibodies specific for the proteins. Lastly, significant class IIMHC-restricted, CD4⁺ T cell reactivity is elicited by GPI-anchoredproteins. Thus GPI anchored proteins appear to be excellent targets forstimulation of protective antibody, Th1-biased CD4⁺ T cell responses,and CD8⁺ T cell responses.

Types of Vaccine-Induced Immunity to T. cruzi

Vaccine-induced immunity to T. cruzi according to the present inventioncan take a variety of fowls. In one embodiment, the vaccine inducessterilizing immunity against T. cruzi in the mammalian host.“Sterilizing immunity” means that a vaccinated, pathogen-free mammalwill, when exposed to the pathogen, not develop a persistent infectionbut instead will totally clear the pathogen (prophylactic vaccination);and also that a pathogen-infected mammal will clear the pathogen and befree of the infection and disease following administration of thevaccine (therapeutic vaccination). However, because a highpercentage—well over 50%—of people infected with T. cruzi fail todevelop chronic disease symptoms even though they appear to remaininfected for their entire lives, it is expected based on the results inthe mouse model reported in the Examples, below, that a balance can bereached in an infected host between an effective immune response andparasite persistence without the development of clinical disease. Thus,in another embodiment, the vaccine elicits a set of responses that aresufficient to delay or, preferably, prevent disease development in T.cruzi infected individuals despite the persistence of parasites. Like avaccine that induces “sterilizing immunity,” this vaccine can beadministered prophylactically, in advance of infection, ortherapeutically, after infection but before the development of a chronicdebilitating disease state. This embodiment of the vaccine is suitablefor delivery to individuals who are infected and at risk of developingdisease.

Prophylactic and Therapeutic Immunization

Accordingly, in a preferred embodiment, the present invention isdirected to both prophylactic and therapeutic immunization against T.cruzi infection and the chronic disease state, known as Chagas' disease,that often eventually follows initial T. cruzi infection. Therapeuticadministration of the polynucleotide or polypeptide vaccine to infectedsubjects is effective to delay or prevent the progression of the T.cruzi infection to a chronic disease state, and also to arrest or curethe chronic disease state that follows T. cruzi infection. Prophylacticadministration of the polynucleotide or polypeptide vaccine touninfected subjects is effective to reduce either or both if themorbidity and mortality associated with infection by T. cruzi. Further,if an uninfected, vaccinated subject is subsequently infected with T.cruzi, the vaccine is effective to prevent progression of the initialinfection to a chronic disease state. As discussed in more detailhereinbelow, the vaccine can contain or encode a single immunogenicpolypeptide or multiple immunogenic polypeptides.

In another preferred embodiment, the invention is directed totherapeutic immunization against other protozoans using a polynucleotideor polypeptide vaccine that preferably stimulates an antibody response,a cell-mediated CD4⁺ immune response and a CD8⁺ immune response. Thevaccine is administered to a mammal infected with a protozoan in theform of a persistent intracellular presence or state. It is contemplatedthat the vaccine can cause the mammal to either clear the parasite(thereby effecting a “cure”), or at least arrest development of disease,thereby preventing or delaying progression of the disease to a chronicdebilitating state. For example, the multicomponent vaccine of theinvention is expected to be effective against Leishmania, Toxoplasma,Eimeria, Neospora, Cyclospora and Cryptosporidia as well as T. cruzi. Itis to be understood that the immunogenic polypeptides used in thevaccine, or the nucleotide sequences encoding them, are derived from theprotozoan against which the vaccine is directed. Methods for identifyingnucleotide sequences encoding such polypeptides from a protozoan genomiclibrary using, for example, expression library immunization (ELI) or DNAmicroarray analysis are described below for T. cruzi but are equallyapplicable to other protozoans.

Advantages of a Genetic Vaccine

The choice of polynucleotide delivery as an immunization techniqueoffers several advantages over other vaccine or antigen deliverysystems. Vaccines containing genetic material are favored overtraditional vaccines because of the ease of construction and productionof the vectors, the potential for modification of the sequences bysite-directed mutagenesis to enhance the antigenic potency of theindividual epitopes or to abolish epitopes that may trigger unwantedresponse, in the case of DNA vaccines, the stability of DNA, the lack ofthe dangers associated with live and attenuated vaccines, their abilityto induce both humoral and cell mediated immunity and, in particular,CD8⁺T cell responses, and the persistence of the immune responses.Successful induction of humoral and/or cellular immune responses toplasmid-encoded antigens using various routes of gene delivery have beenshown to provide partial or complete protection against numerousinfectious agents including influenza virus, bovine herpes virus I,human hepatitis B virus, human immunodeficiency virus-1, as well asparasitic protozoans like Plasmodium and Leishmania (J. Donnelly et al.,Ann. Rev. Immunol. 15:617-648 (1997)). Representative papers describingthe use of DNA vaccines in humans and primates include V. Endresz et al.(Vaccine 17:50-58 (1999)), M. McCluskie et al. (Mol. Med. 5:287-300(1999)), R. Wang et al. (Infect. Immun. 66:4193-202 (1998)), S. LeBorgne et al. (Virology 240:304-315 (1998)), C. Tacket et al. (Vaccine17:2826-9 (1999)), T. Jones et al. (Vaccine 17:3065-71 (1999)) and R.Wang et al. (Science 282(5388):476-80 (1998)). The ability to enhancethe immune response by the co-delivery of genes encoding cytokines isalso well-established.

Polynucleotide Vaccine

The polynucleotide vaccine of the invention includes at least one,preferably at least two, nucleotide coding regions, each coding regionencoding an immunogenic polypeptide component from T. cruzi. When itcontains two or more nucleotide coding regions, the polynucleotidevaccine is referred to herein as a “multicomponent” polynucleotidevaccine. It is desirable to minimize the number of different immunogenicpolypeptides encoded by the nucleotide coding regions in thepolynucleotide vaccine; however, it is nonetheless contemplated that apolynucleotide vaccine that generates the highest level of protectionwill encode 10 or more immunogenic polypeptides. The polynucleotidevaccine can contain DNA, RNA, a modified nucleic acid, or anycombination thereof. Preferably, the vaccine comprises one or morecloning or expression vectors; more preferably, the vaccine comprises aplurality of expression vectors each capable of autonomous expression ofa nucleotide coding region in a mammalian cell to produce at least oneimmunogenic polypeptide or cytokine, as further described below. Anexpression vector preferably includes a eukaryotic promoter sequence,more preferably the nucleotide sequence of a strong eukaryotic promoter,operably linked to one or more coding regions. A promoter is a DNAfragment that acts as a regulatory signal and binds RNA polymerase in acell to initiate transcription of a downstream (3′ direction) codingsequence; transcription is the formation of an RNA chain in accordancewith the genetic information contained in the DNA. A promoter is“operably linked” to a nucleic acid sequence if it is does, or can beused to, control or regulate transcription of that nucleic acidsequence. The invention is not limited by the use of any particulareukaryotic promoter, and a wide variety are known; preferably, however,the expression vector contains a CMV or RSV promoter. The promoter canbe, but need not be, heterologous with respect to the host cell. Thepromoter used is preferably a constitutive promoter.

A vector useful in the present invention can be circular or linear,single-stranded or double stranded and can be a plasmid, cosmid, orepisome but is preferably a plasmid. In a preferred embodiment, eachnucleotide coding region (whether it encodes an immunogenic polypeptideor a cytokine) is on a separate vector; however, it is to be understoodthat one or more coding regions can be present on a single vector, andthese coding regions can be under the control of a single or multiplepromoters.

There are numerous plasmids known to those of ordinary skill in the artuseful for the production of polynucleotide vaccines. Preferredembodiments of the polynucleotide vaccine of the invention employconstructs using the plasmids VR1012 (Vicat Inc., San Diego Calif.),pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad, Calif.) as the vector. Plasmids VR1012 andpCMVI.UBF3/2 are particularly preferred. In addition, the vectorconstruct can contain immunostimulatory sequences (ISS), such asunmethylated dCpG motifs, that stimulate the animal's immune system.Other possible additions to the polynucleotide vaccine constructsinclude nucleotide sequences encoding cytokines, such as granulocytemacrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12)and co-stimulatory molecules such B7-1, B7-2, CD40. The cytokines can beused in various combinations to fine-tune the response of the animal'simmune system, including both antibody and cytotoxic T lymphocyteresponses, to bring out the specific level of response needed to controlor eliminate the T. cruzi infection. The polynucleotide vaccine can alsoencode a fusion product containing the antigenic polypeptide and amolecule, such as CTLA-4, that directs the fusion product toantigen-presenting cells inside the host. Plasmid DNA can also bedelivered using attenuated bacteria as delivery system, a method that issuitable for DNA vaccines that are administered orally. Bacteria aretransformed with an independently replicating plasmid, which becomesreleased into the host cell cytoplasm following the death of theattenuated bacterium in the host cell. An alternative approach todelivering the polynucleotide to an animal involves the use of a viralor bacterial vector. Examples of suitable viral vectors includeadenovirus, polio virus, pox viruses such as vaccinia, canary pox, andfowl pox, herpes viruses, including catfish herpes virus,adenovirus-associated vector, and retroviruses. Exemplary bacterialvectors include attenuated forms of Salmonella, Shigella, Edwardsiellaictaluri, Yersinia ruckerii, and Listeria monocytogenes. Preferably, thepolynucleotide is a vector, such as a plasmid, that is capable ofautologous expression of the nucleotide sequence encoding theimmunogenic polypeptide.

Plasmids and other delivery systems are made using techniques well-knownin the art of molecular biology, as exemplified in the followingexamples. The invention should be understood as including methods ofmaking and using the polynucleotide vaccine.

Polypeptide Vaccine

The polypeptide vaccine of the invention includes at least one,preferably at least two, immunogenic polypeptides from T. cruzi. As withthe polynucleotide vaccine, it is desirable to minimize the number ofdifferent immunogenic polypeptides supplied in the vaccine; however, itis nonetheless contemplated that a polypepetide vaccine that generatesthe highest level of protection will contain 10 or more immunogenicpolypeptides.

Because a CD8⁺ T cell response cannot normally be directly triggered bythe administration of a conventional protein subunit vaccine, theimmunogenic polypeptides contained in the polypeptide vaccine preferablyinclude one or more membrane transporting sequences (MTS) fused to theirN-terminus or C-terminus or both. A membrane transporting sequenceallows for transport of the immunogenic polypeptide across a lipidbilayer, allowing it to be delivered to the inside of a mammalian cell.In a particularly preferred embodiment, the immunogenic polypeptides areshocked with urea, as described further in Example VIII, prior toadministration as a vaccine. From there, portions of the polypeptide canbe degraded in the proteasome, and the resulting peptides can bedisplayed as class I MHC-peptide complexes on the cell surface. In thisway, a polypeptide vaccine can stimulate a CD8⁺ T cell immune response.A polypeptide vaccine of the invention is optionally adjuvanted usingany convenient and effective adjuvant, as known to one of skill in theart.

The invention should be understood as including methods of making andusing the polypeptide vaccine.

Immunogenic Polypeptide

An “immunogenic polypeptide” from a protozoan is a polypeptide derivedfrom the protozoan that elicits in a mammalian host an antibody-mediatedimmune response (i.e., a “B cell” response or humoral immunity), acell-mediated immune response (i.e., a “T cell” response), or acombination thereof. A cell-mediated response can involve themobilization helper T cells, cytotoxic T-lymphocytes (CTLs), or both.Preferably, an immunogenic polypeptide elicits one or more of anantibody-mediated response, a CD4⁺ Th1-mediated response (Th1: type 1helper T cell), and a CD8⁺ T cell response. It should be understood thatthe term “polypeptide” as used herein refers to a polymer of amino acidsand does not refer to a specific length of a polymer of amino acids.Thus, for example, the terms peptide, oligopeptide, and protein areincluded within the definition of polypeptide.

An immunogenic polypeptide for use in a T. cruzi vaccine according tothe invention can be one that is expressed by T. cruzi in theextracellular (trypomastigote) stage, in the intracellular (amastigote)stage, or during both stages of the life cycle. Preferably, theimmunogenic polypeptide is expressed by a T. cruzi amastigote; morepreferably, it is expressed by the amastigote in the early stage ofinfection, within about 24 hours from initial infection. Alsopreferably, the immunogenic polypeptide is a membrane surface-associatedpolypeptide or a secreted polypeptide. Surface associated-immunogenicpolypeptides include, for example, T. cruzi proteins that are anchoredto the plasma membrane by glycosylphosphotidylinositols, or GPIs, andthose that have transmembrane domains or are otherwise embedded in theplasma membrane. One class of polypeptides that exemplifies immunogenicpolypeptides according to the invention is the trans-sialidase family ofproteins, such as TSA-1 (T. cruzi Peru; D. Fouts et al., Mol. Biochem.Parasitol. 46:189-200 (1991); GenBank Acc. Number M58466), ASP-1 andASP-2 (T. cruzi Brazil; M. Santos et al., Mol. Biochem. Parasitol.86:1-11 (1997); GenBank Ace. Number U74494)) and ASP-2 (T. cruzi Brazil;H. Low et al., Mol. Biochem. Parasitol. 88:137-149 (1997); GenBank Acc.Number U77951), which are found in both secreted and surface-displayedforms; other examples are proteins that are secreted upon entry of thehost cells by T. cruzi, such as the hemolysin, and the Lyt1 protein(porin).

In a preferred embodiment, the polynucleotide vaccine of the inventioncontains at least one nucleotide coding region that encodes animmunogenic polypeptide that constitutes in an amastigote-expressed CD8⁺T cell target molecule. Analogously, the polypeptide vaccine of theinvention contains at least one immunogenic polypeptide that constitutesin an amastigote-expressed CD8⁺ T cell target molecule As noted above,the induction of T helper cell responses skewed toward the production oftype 1 cytokines such as IL-2 and IFN-γ, which presumably potentiateantibody responses and macrophage activation, is also of substantialimportance in immunity to T. cruzi, as is antibody production. Thus, ina particularly preferred embodiment, the vaccine of the inventioncontains a plurality of immunogenic polypeptides (or, in the case of apolynucleotide vaccine, a plurality of nucleotide coding regionsencoding immunogenic polypeptides), such that in combination theimmunogenic polypeptides stimulate all three immune responses identifiedherein, namely a protective antibody response, a Th1-biased CD4⁺ T cellresponse, and a CD8⁺ T cell responses.

An immunogenic polypeptide used in the compositions of the invention isnot limited to a naturally occurring immunogenic protozoan polypeptide;it can include an immunogenic fragment or immunogenic analog of aprotozoan polypeptide. Likewise the immunogenic polypeptide can be amultivalent polypepdtide that has been engineered to include epitopesobtained from different immunogenic polypeptides of the protozoan. Animmunogenic analog of an immunogenic protozoan polypeptide is apolypeptide having one or more amino acid substitutions, insertions, ordeletions relative to an immunogenic protozoan polypeptide, such thatimmunogenicity is not entirely eliminated. Substitutes for an amino acidare preferably conservative and are selected from other members of theclass to which the amino acid belongs. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and tyrosine. Polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine, asparagineand glutamine. The positively charged (basic) amino acids includearginine, lysine and histidine. The negatively charged (acidic) aminoacids include aspartic acid and glutamic acid. Examples of preferredconservative substitutions include Lys for Arg and vice versa tomaintain a positive charge; Glu for Asp and vice versa to maintain anegative charge; Ser for Thr so that a free —OH is maintained; and Glnfor Asn to maintain a free NH₂. Immunogenic fragments of an immunogenicprotozoan polypeptide are immunogenic protozoan polypeptides that aretruncated at either or both of the N-terminus or C-terminus, withouteliminating immunogenicity. Preferably, an immunogenic fragment containsan epitope recognized by a host T cell. Fragments of an immunogenic T.cruzi protein contain at least about eight amino acids, preferably atleast about 12 amino acids, more preferably at least about 20 aminoacids.

Cytokines

Preferably, the polynucleotide vaccine further includes at least onenucleotide coding region encoding a cytokine. Preferred cytokinesinclude interleukin-12 (IL-12), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-18(IL-18), γ-interferon, α,β-interferons, and chemokines. Especiallypreferred cytokines include 1′-12 and GM-CSF.

Pharmaceutical Compositions

The polynucleotide and polypeptide vaccines of the invention are readilyformulated as pharmaceutical compositions for veterinary or human use.The pharmaceutical composition optionally includes excipients ordiluents that are pharmaceutically acceptable as carriers and compatiblewith the genetic material. The term “pharmaceutically acceptablecarrier” refers to a carriers) that is “acceptable” in the sense ofbeing compatible with the other ingredients of a composition and notdeleterious to the recipient thereof. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine may containminor amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, salts, and/or adjuvants which enhance theeffectiveness of the immune-stimulating composition. Methods of makingand using such pharmaceutical compositions are also included in theinvention.

Administration of the Polynucleotide Vaccine

The polynucleotide vaccine of the invention can be administered to themammal using any convenient method, such as intramuscular injection,topical or transdelinal application to the mammal's skin, or use of agene gun, wherein particles coated with the polynucleotide vaccine areshot into the mammal's skin. The amount of polynucleotide administeredto the mammal is affected by the nature, size and disease state of themammal as well as the delivery method; for example, typically less DNAis required for gene gun administration than for intramuscularinjection. Further, if a polynucleotide encoding a cytokine isco-delivered with nucleotide coding regions encoding the immunogenicpolypeptide from T. cruzi, the amount of polynucleotide encoding theimmunogenic polypeptide from T. cruzi in the vaccine is optionallyreduced.

Hundreds of publications have now reported the efficacy of DNA vaccinesin small and large animal models of infectious diseases, cancer andautoimmune diseases (J. Donnelly et al., Rev. Immunol. 15:617 (1997).Vaccine dosages for humans can be readily extended from the murinemodels by one skilled in the art of genetic immunization, and asubstantial literature on genetic immunization of humans is nowavailable to the skilled practitioner. For example, Wang et al. (Science282:476-480 (1998)) vaccinated humans with plasmid DNA encoding amalaria protein, and the same group has developed a plan formanufacturing and testing the efficacy of a multigene Plasmodiumfalciparum liver-stage DNA vaccine in humans (Hoffman et al., Immunol.Cell Biol. 75:376 (1997)). In general, the polynucleotide vaccine of theinvention is administered in dosages that contain the smallest amount ofpolynucleotide necessary for effective immunization. It is typicallyadministered to human subjects in dosages containing about 20 μg toabout 2500 μg plasmid DNA; in some instances 500 μg or more of plasmidDNA may be indicated. Typically the vaccine is administered in two ormore injections at time intervals, for example at four week intervals.

Administration of the Polypeptide Vaccine

Like the polynucleotide vaccine, the polypeptide vaccine can beadministered to the mammal using any convenient method, such asintramuscular or intraperitoneal injection, topical administration, oralor intranasal administration, inhalation, perfusion and the like. Theamount of polypeptide administered to the mammal is affected by thenature, size and disease state of the mammal, as well as by the deliverymethod. Intraperitoneal injection of 25 to 50 ug of polypeptidecontaining a membrane transducing sequence has been shown to result inimport of the protein into nearly 100% of murine blood and spleen cellswithin 20 minutes (Schwarze et al., Science 285:1569-1572 (1999)) andthe sensitization of cytotoxic T cells (M.-P. Schutze-Redelmeier et al.,J. Immunol. 157:650-655 (1996)). Useful dosages of the polypeptidevaccine for humans can be readily determined by evaluating its activityin vivo activity in mice.

Administration of a Combination of Polynucleotide Vaccine andPolypeptide Vaccine

The invention contemplates administration of both a polynucleotidevaccine and a polypeptide vaccine to a mammal in a serial protocol. Forexample, a plasmid-based DNA vaccine may be administered to a mammal to“prime” the immune system, followed by the one or more administrationsof a polypeptide vaccine or a viral vaccine (e.g., vaccinia vectorcarrying the genes that encode the immunogenic polypeptides and,optionally, cytokines) to further stimulate the mammal's immune system.The order of administration of the different types of vaccines, and thenature of the vaccines administered in any given dose (e.g., polypeptidevaccine, plasmid vaccine, viral vector vaccine) can be readilydetermined by one of skill in the art to invoke the most effectiveimmune response in the mammal.

Methods of Screening for T. cruzi Nucleotide Sequences EncodingCandidate Immunogenic Polypeptides

As noted above, the polynucleotide vaccine of the invention can includeone or more nucleotide coding regions encoding a polypeptide from thetrans-sialidase family of proteins, such as TSA-1, ASP-1 and ASP-2, or apolypeptide that is secreted upon entry of the host cells by T. cruzi,such as the hemolysin or Lyt1 protein. Likewise, the polypeptide vaccineof the invention can contain one or more of these polypeptides. It iscontemplated that other polypeptides, as yet unidentified, may also beincluded in (or encoded by nucleotide sequences included in) thesevaccines in order to produce the desired type and level of immuneresponse in the mammal. To identify candidate polypeptides, theinvention provides a genome-based method for evaluating the ability ofprotozoan polypeptides to stimulate a mammal's immune response.

The classical approach to vaccine development, particularly withparasites, has been to use somewhat rational approaches to identifypotential vaccine candidates, clone the genes and express the candidateproteins, and finally to test the ability of the proteins to induce aneffective immune response. This approach has a number of seriousdrawbacks. First, the rational identification of candidates is notalways so easy to do “rationally.” Second, this approach makesassumptions about the type of immune response that should be protective,sometimes on the basis of not so firm data. Third it is extremely timeconsuming and with no real guarantee that at the end of the 3-5 years itmight take to accomplish, the single candidate which is the focus of thework will be even marginally effective as a vaccine. Fourth, it isexpensive. And fifth, in many cases the proteins cannot be delivered ina way that will generate sufficient and appropriate (i.e. protective)immune responses.

The novel method for identifying additional immunogenic polypeptidesthat is provided by the present invention relies upon genome-basedapproaches that utilize both rational and unbiased techniques forvaccine discovery and genetic immunization for vaccine delivery. Theseapproaches integrate the current understanding of immunity and diseasein protozoan infection with, in the case of T. cruzi, informationemerging from genome projects with trypanosomitids, as well as newtechnical developments for genome analysis and vaccine delivery, whilecontinuing to make use of the strength of the mouse model of protozoaninfection.

In one embodiment of the method for screening for vaccine candidates inT. cruzi, the invention utilizes expression library immunization (ELI)(M. Barry et al, Nature. 377:632-5 (1995). ELI provides an unbiasedmethod (i.e. makes no assumptions about what types of molecules or whattypes of immune responses are necessary for protection) for screeningpotentially the entire genome of a protozoan. A genetic immunizationtechnique is used to select protective antigen encoding genes from thepathogen DNA. For this, a eukaryotic expression library of the pathogenDNA is constructed in a genetic immunization vector. Animals areimmunized with fractions of the library, and then monitored for theinduction of immune responses and protection from challenge infection.Sub-groups of the genomic library which provide protection are then“sibbed” in stages into smaller fractions and tested for elicitation ofprotective immune responses and resistance to infection. A series ofimmunization/challenge studies are done until individual protectivegenes are identified. Advantages associated with ELI include: (i)instead of cloning gene by gene and testing the immunogenic propertiesof each gene product, ELI screens the whole genome and selects for theprotective genes, (ii) candidate genes are selected by ELI based uponthe immune system itself, therefore knowledge of the parasite biology orthe immunogenic properties of individual antigens is not required, (iii)ELI is not only a cheaper, faster and more effective method for genediscovery, it might prove to be the only avenue for the generation of aneffective vaccine for pathogens like T. cruzi which are difficult togrow, maintain and attenuate for their use as vaccine, and (iv) ELIutilizes a vaccine delivery method for the discovery of immunogenicgenes. Conveniently, with the identification of new genes which can beused to constitute the anti-T. cruzi genetic vaccine, the vaccinedelivery method is established as well.

Another embodiment of the screening method of the invention utilizes DNAmicroarrays. DNA microarrays provide a tool to monitor the expression ofmany (or all, if one chooses) of the genes in the genome of a protozoan.Genes whose expression is upregulated in the parasite soon afterinfection of the host cell are identified, since the protein products ofsuch genes would be among the first to reach the class I MHC processingand presentation pathway and thus the early indicators to the immunesystem of the infection status of host cells.

Vaccine candidates are tested singly and in combination under variousconditions in murine models for protection of mice from both lethalinfection and from development of severe disease. Whether or not thesemolecules are recognized by humans infected with the protozoan can bereadily determined using ELISA and CTL assays as described in thefollowing examples.

Murine Model for Chagas' Disease

The most widely characterized animal model for Chagas' disease is themouse. The mouse model parallels human infection in that 1) both acuteand chronic stages of infection are generally present, 2) recovery fromthe acute infection is dependent on the development of a non-sterilizingimmune response, 3) chronically infected mice develop pathological andelectrocardiographic changes similar to those seen in human Chagas'disease and 4) many of the immune mechanisms thought to be important inimmune control of the parasite in humans are also present in the mousemodel. During T. cruzi infection, both chagasic patients andexperimental animals produce strong immune responses to moleculesexpressed in the infective non-replicative trypomastigote stage and thereplicative amastigote forms (N. Andrews et al., Am. J. Trop. Med. Hyg.40:46-9 (1989); A. Krettli, J. Immunol. 128:2009-2012 (1982)). A WorldHealth Organization (WHO) working group concluded that the mouse bestmodel system for studying the various aspects of Chagas' disease (WHOReport, 1984).

Genetic immunization has been demonstrated herein for BalbC mice (singlecomponent genetic vaccine, TSA-1) and B6 mice (multiple componentgenetic vaccine, TSA-1, ASP-1 and ASP-2) using infection with Brazil T.cruzi. Standard polynucleotide vaccine dosages for mice range from 1 ngto 100 μg of polynucleotide. Both murine systems make good human modelssince initial infection with T. cruzi is not generally lethal, and achronic disease state typically develops following initial infection.Prophylactic genetic immunization was also tested in C3H mice (multiplecomponent genetic vaccine), but with less promising results. However,C3H mice represent a murine mode that is extremely susceptible toinfection with T. cruzi (typically 100% mortality is observed uponinitial infection) and thus is not as representative of T. cruziinfection and subsequent disease progression in humans.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example I Vaccination with Trypomastigote Surface Antigen-1-encodingPlasmid DNA Confers Protection Against Lethal Trypanosoma cruziInfection

Mice and parasites. Six- to 8-wk-old female C57BL/6J (B6) and BALB/cByJ(BALB/c) mice (breeding pairs obtained from The Jackson Laboratory (BarHarbor, Me.) were used in all experiments. The Brazil strain of T. cruziwas maintained in vivo by serial biweekly passage of 10³ blood-formtrypomastigotes (BFT) in C3H/HeSnJ mice and by continuous in vitropassages of tissue culture-derived trypomastigotes (TCT) in monolayersof Vero cells. B6 mice were infected intraperitoneally with 10³ BFT andchallenged 3 months later challenged with 10⁵ TCT by subcutaneousinjection at the base of the tail.

Cell lines and culture reagents. P815 (H-2^(d); mastocytoma cells; ATCCTIB 64), J774 (H-2^(d), macrophages, ATCC TIB 67), 3T3 (H-2^(d),fibroblasts, ATCC CCL 163), Vero (African Green monkey kidney cells,ATCC CCL 81) (all from American Type Culture Collection [ATCC],Rockville, Md.), RMA-S (peptide TAP.2 transporter-deficient, low H-2^(b)expressor mutant of the RBL-5 Rauscher virus-induced T cell lymphoma;provided by Dr. H.-G. Ljundggren, Karolinska Institute, Stockholm,Sweden) and 5A.Kb. 3 cells (H-2^(k) fibroblasts stably transfected withthe Kb gene; provided by Dr. S. Jameson, University of Minnesota,Minneapolis, Minn.) were maintained in complete RPMI-1640 (Mediatech,Herndon, Va.) medium (CR) containing 10% heat-inactivated fetal bovineserum (HyClone, Logan, Utah), 20 mM HEPES, 2 mM L-glutamine, 1 mM sodiumpyruvate, 0.1 mM non-essential amino acids and 50 μg/ml gentamicin (allfrom Gibco BRL, Gaithersburg, Md.). COS-7 cells (SV40 transformedAfrican Green monkey kidney cells; ATCC CRL 1651) were grown insimilarly supplemented Dulbecco's modified Eagle's medium (Mediatech,Herndon, Va.). T cell medium (TCM) was prepared by supplementing CR with50 μM 2-mercaptoethanol (Gibco BRL).

Peptides. The peptide TSA-1₅₁₅₋₅₂₂ (VDYNFTIV; SEQ ID NO:1), representingthe H-2K^(b)-restricted T. cruzi Trypomastigote Surface Antigen-1 CTLepitope was produced using Fmoc-based solid phase chemistry on an ACTMPS 350 peptide synthesizer (Advanced Chem Tech, Louisville, Ky.) byMolecular and Genetic Instrumentation Facility at University of Georgia(Athens, Ga.). The H-2 K^(b)-restricted OVA CTL peptide OVA₂₅₇₋₂₆₄(SIINFEKL; SEQ ID NO:2) was used as a control. Lyophilized peptides weredissolved at 20 mg/ml in DMSO and stored at −70° C. Before use, peptideswere diluted with RPMI-1640. Peptides were not toxic to target cells oreffector cell cultures.

Plasmid DNA constructs. The genomic DNA fragments of the TSA-1 gene (D.Fouts et al., Mol Biochem Parasitol. 46:189-200 (1991); D. Peterson etal., Nature 322:566-8 (1986)) encoding amino acid residues 78 to 652 and78 to 790, excluding and including, respectively, the 5 nonapeptidetandem repeat unit, were amplified by PCR using pBluescript II SK(+)/TSA-1 (provided by Dr. David Fouts, University of California,Irvine, Calif.) as a template. Forward and reverse primers were designedto incorporate, respectively, SalI and XbaI restriction sites(underlined below) for directional cloning. Primers were constructed onan Applied Biosystems 394 DNA/RNA synthesizer (Foster City, Calif.) atthe Molecular Genetics Instrumentation Facility. The forwardoligonucleotide primer 5′-AGTCGACGGATCCATGATTGCATTTGTCGAAGGC-3′ (SEQ IDNO:3) was used with reverse primers5′-ATCTAGAAGCTTCATAGTTCACCGACACTCAGTGG-3′ (SEQ ID NO:4) and5′-ATCTAGAAGCTTCATGCCGCAGCATTTGCTTCCCC-3′ (SEQ ID NO:5), to amplify a1.7 kb (repeatless TSA-1₇₈₋₆₅₂) and a 2.1 kb (repeat-bearingTSA-1₇₈₋₇₉₀) product, respectively. The amplification productscontaining the A overhangs generated by Taq DNA polymerase during thePCR reaction were cloned into the HindIII site of pUC19-T vector.Following digestion with SalI and XbaI, the 1.7 kb and 2.1 kb TSA-1fragments were gel purified and cloned into the SalI and XbaI sites ofthe eukaryotic expression vector VR1012 (Vical Inc., San Diego, Calif.)G. Hartikka et al., Hum. Gene Ther. 7:1205-1217 (1996)) to generateVR1012 TSA1.7 and VR1012 TSA2.1. In the VR1012 vector, expression of theencoded gene is driven by a CMV immediate-early gene promoter.Constructs were transformed into Escherichia coli DH5-competent cellsand grown in Luria Bertani broth with 70 μg/ml kanamycin. Closedcircular plasmid DNA was purified by anion exchange chromatography usingthe Qiagen maxi prep kit (Qiagen, Chatsworth, Calif.) according tomanufacturer's specifications. Plasmid DNA was sterilized by ethanolprecipitation and dissolved in sterile phosphate-buffered saline (PBS).

In vitro expression. Expression of the VR1012 TSA1.7 and VR1012 TSA2.1in COS-7 cells was assessed in vitro by transient transfection. COS-7cells were seeded in 6-well plates (Costar, Cambridge, Mass.) at 2×10⁵cells/well in 3 ml complete DMEM and incubated overnight at 37° C., 6%CO₂. In a final volume of 300 μl, 10 μg of plasmid DNA (unmodifiedVR1012 plasmid, TSA-1-encoding VR1012 TSA1.7 or VR1012 TSA2.1) weremixed with 30 μg of Lipofectin Reagent (Gibco BRL), and the mixtureincubated for 15 minutes at room temperature before being diluted with1.7 ml serum-free MEM. After washing COS-7 monolayers (50-70% confluent)with serum-free MEM, cells were overlayed with the mixture containingthe DNA-Lipofectin complexes and incubated overnight at 37° C., 6% CO₂.Cell culture media was then replaced with 3 ml complete DMEM andincubated for an additional day. Transiently transfected COS-7 cellswere harvested by gentle trypsinization, washed in PBS, and seeded in8-well Lab Tek chamber slides (Nunc Inc., Naperville, Ill.) at 1×10⁴cells/well. After overnight incubation at 37° C., 6% CO₂, cells werewashed with PBS, fixed in ice cold methanol for 15 minutes at 4° C. andwashed 4 more times before blocking with PBS-1% bovine serum albumin(BSA) for 1 hour at 37° C. Cells were subsequently stained for 2 hoursat 37° C. with a polyclonal anti-T. cruzi serum obtained from acutelyinfected C3H/HeSnJ mice or with normal mouse serum (1:200 dilution inPBS-1% BSA), washed 3 times, and finally incubated for 1 hour at roomtemperature with fluorescence isothiocyanate (FITC)-labeled F(ab)₂ goatanti-mouse IgG (1:50 dilution in PBS-1% BSA) (Southern Biotechnology,Birmingham, Ala.). Slides were then rinsed 4 times with PBS-1% BSA andmounted in 10% glycerol, 0.1 M sodium bicarbonate (pH 9), 2.5%1,4-diazobicyclo-(2,2,2) octane for visualization by laser scanningconfocal microscopy (MRC-600) (Bio-Rad Laboratories, Hercules, Calif.).

Genetic immunizations and challenges. Groups of B6 and BALB/c mice wereinjected intramuscularly into each tibialis anterior muscle with 50 μgof VR1012 TSA1.7, VR1012 TSA2.1 or control VR1012 suspended in 50 μl PBSusing a 27-gauge needle. Mice were boosted 4 weeks later with anidentical dose of plasmid (100 μg total) given by the same bilateralintramuscular injection. Tail blood samples were collected 3 and 2 weeksafter the first and second dose, respectively, and sera stored at −20°C. until assayed for anti-T. cruzi antibody. Two weeks after the seconddose, animals were infected by intraperitoneal injection of 10⁵ (B6) or10³ (BALB/c) T. cruzi BFT. Parasitemias were monitored periodicallyusing hemacytometer counts of 10 μl tail-vein blood in an ammoniumchloride solution. Mortality was recorded daily.

Determination of serum antibody levels. Antibody responses induced bythe immunization of mice with plasmid DNA was evaluated by a solid-phaseenzyme-linked Immunosorbant assay (ELISA). In brief, capture antigen wasprepared by sonication of PBS-washed 5×10⁷ T. cruzi parasites (80%trypomastigotes; 20% amastigotes) in 50 mM carbonate-bicarbonate buffer(pH 9.6). Sonicated material was spun for 1 hour at 100,000×g at 4° C.Wells of flexible polyvinyl chloride 96-well plates (Falcon, BectonDickinson & Co., Oxnard, Calif.) were coated overnight at 4° C. with 100μl of a predetermined optimal dilution (5×10⁵ parasites/well) of thesoluble antigen. Washed wells were blocked with 1% BSA in PBS-0.05%Tween 20 (PBST) for 1 hour at 37° C. After blocking, 100 μl of pooledmouse sera (1:100 dilution in PBST) was added to the plates andincubated for 1 hour at 37° C. Plates were washed 6 times with PBST andincubated for an additional hour with 100 μl of a horseradishperoxidase-labeled goat anti-mouse immunoglobulin (A, G, M) (1:1000dilution in PBST) (Cappel, Organon Teknika Corp., West Chester, Pa.).Washed wells were developed with 100 μl of the substrate2,2′-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) and absorbanceread at 405 nm using an automated ELISA microplate reader (Bio-TekInstruments, Winooski, Vt.).

Generation of effector cells. Unless otherwise indicated, spleens fromDNA-immunized mice were removed two weeks after the last dose and immunespleen cell (SC) suspensions prepared in TCM. In the case of B6 mice,spleen cells (SC) were cultured in 24-well plates at 5×10⁶ cells/well.TSA-1₅₁₅₋₅₂₂ peptide was included in each 2-ml culture at 1 μM finalconcentration. In the case of BALB/c mice, 35×10⁶ SC in 10 ml TCM werecultured in upright 25-cm² tissue culture flasks containing irradiatedmonolayers of stimulator T. cruzi-infected J774 cells. After 2 days ofincubation at 37° C., 6% CO₂, cultures were made to 5% Rat T-STIMwithout Con A (Collaborative Biomedical Products, Bedford, Mass.) andincubated for 4 additional days. Effector cells from BALB/c mice werealso unstimulated immune SC without undergoing secondary in vitrostimulation. SC from B6 mice chronically infected with T. cruzi wereobtained 6 months after parasite challenge and stimulated as describedfor SC from DNA-immunized animals.

Preparation of Peptide-Pulsed Target Cells. Peptide-Pulsed Targets wereused to measure CTL activity of peptide-stimulated effector cellsgenerated from plasmid DNA-immunized B6 mice. RMA-S(H-2^(b)) cellspreincubated for 24 h at 26° C., 6% CO₂, were seeded into 24-well plates(Costar, Cambridge, Mass.) at 10⁶ cells/well in 2 ml CR and incubatedovernight under the same conditions in the presence of 0.05 μm ofISA-1₅₁₅₋₅₂₂ peptide or OVA₂₅₇₋₂₆₄ negative control peptide and 100 μCiof a sterile Na₂ ⁵¹CrO₄ solution (⁵¹Cr) (Amersham Life ScienceCorporation, Arlington Heights, Ill.). Two hours prior to theirprocessing for CTL assays, cells were shifted 37° C., 6% CO₂. P815(H-2^(d)) target cells were also prepared in 24-well plates by overnightincubation at 37° C., 6% CO₂, with ⁵¹Cr and TSA-1₅₁₅₋₅₂₂ peptide.

Preparation of T. cruzi-infected stimulator and target cells. T.cruzi-infected cells were used to generate and measure the CTL activityof effector cells from plasmid DNA-immunized BALB/c mice. Monolayers ofJ774 cells (60% confluent) prepared in upright 25-cm² tissue cultureflasks (Corning, N.Y.) were infected overnight with T. cruzi TCT (50:1parasite to host cell ratio). After extensive washing with serum-freeRPMI 1640 to remove noninvading parasites, infected monolayers wereirradiated (14 Krad) (Gammacell 200, ⁶⁰Co source) and then used asstimulators for immune SC. To prepare T. cruzi-infected target cellsused to ascertain the lytic activity of BALB/c-derived stimulated SC,monolayers (50% confluent in horizontal 25-cm² flasks) of MHC-matched3T3 (H-2^(d)) and mismatched 5A.Kb. 3 (H-2^(k) and H-2K^(b)) cells wereincubated for 2 days at 37° C., 6% CO₂, in CR supplemented with 1000U/ml of IFN-+13 (Lee Biomolecular Laboratories, Inc., San Diego,Calif.), washed and then infected overnight with T. cruzi TCT (50:1parasite to host cell ratio). After washing, T. cruzi-infectedmonolayers were treated with PBS-1 mM EDTA to prepare single cellsuspensions and washed once more before a 1-h ⁵¹Cr-labeling step at 37°C. To assess the lytic activity of unstimulated BALB/c-derived immuneSC, monolayers of untreated J774 cells were infected and single cellsuspensions for ⁵¹Cr labeling were prepared by moderate pipetting of thecell monolayer. Under the conditions described, stained (Leukostat,Fisher Scientific, Atlanta, Ga.) cytospin preparations of each cultureindicated that 65-75% of the cells were infected.

CTL assay. Cytolytic activity was measured by the ⁵¹Cr release assay, asdescribed in Wizel et al. (Eur. J. Immunol. 24:1487-95 (1994)). Inbrief, ⁵¹Cr-labeled target cells were washed three times in CR andresuspended in TCM, and 5×10³ targets (100 μl) were added to effectorcells (100 μl) at various effector-cell-to-target-cell (E:T) ratios in96-well round-bottom plates (Corning). After a 5 hour incubation at 37°C., 6% CO₂, supernatants were harvested with the Skatron SCS System(Skatron, Sterling, Va.) and radioactivity counted on a Cobra IIAutogamma counter (Packard Instrument Company, Downers Grove, Ill.).Percent-specific lysis was calculated from the mean of triplicates asfollows: 100×[(experimental release—spontaneous release)/(maximumrelease—spontaneous release)]. Maximum and spontaneous release weredetermined in wells containing no effectors in the presence or absenceof 2% Triton X-100, respectively. In experiments where CTL activity ofCD8⁺ and CD4⁺T cells was tested, effector cells were depleted byincubation on ice for 30 minutes with predetermined dilutions of culturesupernatants from hybridomas 3.155 (anti-CD8) (ATCC TIB 211), and RL172(anti-CD4), followed by 30 minutes at 37° C. in the presence of 1:6diluted rabbit complement (Pel-Freez, Brown Deer, Wis.). Spontaneousrelease did not exceed 20% of the maximum release. SE ranged between0.02 to 6.1% of the mean.

Results

Expression of TSA-1 in transiently transfected cells. To study theeffectiveness of genetic immunization against T. cruzi, the TSA-1 genewas subcloned into the VR1012 mammalian expression vector containing theCMV promoter and the bovine growth hormone polyadenylation sequences.The constructs VR1012 TSA1.7 and VR1012 TSA2.1 were generated to drivethe expression of two N-terminally truncated TSA-1 gene products lackingand bearing, respectively, the 5 nonapeptide repeats located near theC-terminal end of the TSA-1 protein. Both plasmid constructs expressedthe inserted TSA-1 gene fragment upon transient transfection of COS-7cells. The cytoplasmic expression of TSA-1 in VR1012 TSA1.7- and VR1012TSA2.1-transfected cells was intense as detected by immunofluorescentstaining with a polyclonal anti-T cruzi serum. In contrast, similarlytransfected cells stained with normal mouse serum showed no evidence ofimmunofluorescence. No expression was detected in cells transfected withthe unmodified VR1012 vector and stained with either sera.

Immunization with TSA-1 plasmid DNA elicits parasite-specific antibodyresponse. A strong humoral immune response has been widely implicated asa major effector mechanism that participates in the immune control of T.cruzi and immunization of mice with a recombinant N-proximal portion ofTSA-1 induces an antibody response which correlates with survival to alethal challenge infection. To ascertain whether a T. cruzi-specificantibody response could be elicited by the expression of the TSA-1protein fragments following intramuscular DNA immunization, BALB/c andB6 mice were injected with 100 μg VR1012 TSA1.7, VR1012 TSA2.1 orunmodified VR1012 control plasmid (50 μg of split in each tibialisanterior muscle). Mice were boosted after 4 weeks with the same dose ofplasmid. The presence of parasite-specific antibodies in pooled seraprepared from each group of mice was assessed by ELISA (FIG. 1). Threeweeks following the first dose, sera from BALB/c mice immunized witheither VR1012 TSA1.7 or VR1012 TSA2.1 showed comparable antibodyresponses against the sonicated parasite material used as captureantigen. Two weeks after the second dose, while a boosting in theparasite-specific antibody level was detected in the sera from VR1012TSA1.7-immunized group, the level of antibodies in the sera from theVR1012 TSA2.1-immunized animals remained essentially unchanged. When asimilar analysis was conducted for the pooled sera from similarlyimmunized B6 mice, the antibody levels after the first dose did notexceed the level found in normal mouse serum. However, after the seconddose, only the VR1012 TSA2.1-immunized group showed a parasite-specificantibody response. In all cases, the antibody levels detected in thesera from groups of mice immunized with unmodified VR1012 vector was nodifferent than the level measured in normal mouse serum.

Induction of long-lasting TSA-1-specific CTL response in TSA-1 plasmidDNA-immunized B6 mice. TSA-1₅₁₅₋₅₂₂ is a target of H-2 K^(b)-restrictedprotective CTL responses induced in B6 mice infected with T. cruzi. Wetherefore wanted to determine whether immunization of this strain ofmice with the TSA-1-encoding DNA vectors could induce aTSA-1₅₁₅₋₅₂₂-specific CTL response. Two weeks after the secondintramuscular injection of either VR1012 TSA1.7 or VR1012 TSA2.1, immuneSc were stimulated with TSA-1₅₁₅₋₅₂₂ (1 μM). After 6 days, recall CTLactivity of undepleted responder cultures was assessed in a 5 hour ⁵¹Crrelease assay against RMA-S(H-2^(b)) and P815 (H-2^(d)) target cellssensitized with TSA-1₅₁₅₋₅₂₂ peptide (0.05 μM) at the indicated E:Tratios. RMA-S cells pulsed with OVA₂₅₇₋₂₆₄ peptide (0.05 μM) were usedas negative control target cells. CTL activity of effector cellsdepleted of CD4⁺ or CD8⁺T cells was measured at a 50:1 E:T ratio againstTSA-1₅₁₅₋₅₂₂-sensitized (0.05 μM) target cells. CTL activity was antigenspecific, MHC class I-restricted and dependent on CD8⁺T lymphocytes(FIG. 2A). The H-2^(b) effector cells lysed matched RMA-S cells(H-2^(b)) sensitized with TSA-1₅₁₅₋₅₂₂ but were unable to lyse the samecells pulsed with control peptide OVA₂₅₇₋₂₆₄ or MHC-mismatched P815cells (H-2^(d)) pulsed with TSA-1₅₁₅₋₅₂₂.

Detected lytic activity was abrogated by CD8⁺ T cell depletion, but notby depletion of CD4⁺ effectors. In no case did TSA-1₅₁₅₋₅₂₂-stimulatedSC from mice immunized with unmodified VR1012 vector display CTLactivity against peptide-sensitized target cells. SimilarTSA-1₅₁₅₋₅₂₂-specific CTL activity was detected in thepeptide-stimulated SC cultures established 7 mo after mice had receivedthe second 100 μg dose of the TSA-1-encoding DNA vectors (FIG. 2B). Themagnitude of such recall CTL response was comparable to the CTL activitydetected for TSA-1₅₁₅₋₅₂₂-stimulated effectors from T. cruzi-infectedmice. Hence, immunization of B6 mice with both TSA-1-encoding DNAconstructs generates a long-lasting TSA-1₅₁₅₋₅₂₂-specific CTL responsewhich closely resembles the recall response induced in T. cruzi-infectedanimals.

CTL response induced in BALB/c mice by TSA-1 plasmid DNA immunization isparasite-specific, MHC class I-restricted and CD8⁺ T cell-dependent.Despite the fact that the target antigens recognized by CTL from T.cruzi-infected BALB/c mice (H-2^(d)) have not been identified, SC fromthese animals display genetically restricted CTL activity against T.cruzi-infected target cells. Thus, we used this system to determinewhether parasite-specific CTLs could be induced in BALB/c mice followingimmunization with the TSA-1-encoding plasmid DNA constructs. Two weeksafter the second vaccination, immune SC from BALB/c (H-2^(d)) mice wereprepared and tested unstimulated for CTL recognition of T.cruzi-infected or uninfected J774 macrophages (H-2^(d)) and T.cruzi-infected 5A.Kb. 3 fibroblasts (H-2^(k); H-2K^(b)) in a 5-h ⁵¹Crrelease assay at the indicated E:T ratios (FIG. 3A). Following a 6-daystimulation period with irradiated T. cruzi-infected J774 macrophages,effector cells were assayed at the indicated E:T ratios for CTL activityon ⁵¹Cr labeled T. cruzi-infected or uninfected 3T3 fibroblasts(H-2^(d)) and T. cruzi-infected 5A.Kb. 3 cells. Effector cells depletedof CD4⁺ or CD8⁺ T cells were tested for CTL activity at a 50:1 E:T ratioagainst T. cruzi-infected 3T3 cells. Levels of infection in stimulatorcells and target cells ranged from 65 to 75%. Infection of J774 cells(H-2^(d)) with T. cruzi efficiently targeted these macrophages for lysisby the H-2^(d) effector cells harvested from either VR1012 TSA1.7- orVR1012 TSA2.1-immunized mice. In contrast, minimal or no lysis wasdetected against uninfected J774 cells and against mismatched T.cruzi-infected 5A.Kb. 3 fibroblasts (H-2^(k); H-2K^(b)). None of thetarget cells tested was recognized by effector cells obtained fromcontrol VR1012-immunized animals. Then, CTL activity of immune SC thathad been stimulated for 6 days with T. cruzi-infected J774 macrophageswas assessed against uninfected and T. cruzi-infected fibroblasts (FIG.3B). Again, the specificity and MHC class I-restricted nature of therecall CTL response was demonstrated by the ability of effector cellsderived from VR1012 TSA1.7- and VR1012 TSA2.1-immunized mice to lyseinfected but not uninfected 3T3 cells (H-2^(d)) and by their inabilityto recognize infected 5A.Kb. 3 cells (H-2^(k); H-2 Kb). When thephenotype of the VR1012 TSA1.7-derived effectors was tested, it wasfound that they were CD8⁺CD4⁻ because the lytic activity of these cellswas significantly reduced by the depletion of CD8⁺ T cells and minimallyaffected by the depletion of CD4⁺ T cells. Similarly stimulated VR1012immune SC failed to lyse all the target cells tested. Altogether, thesedata indicated that immunization of BALB/c mice with TSA-1-encoding DNAplasmids efficiently primed parasite-specific CD8⁺ CTL precursors andthat these in vivo expanded cells were in sufficient numbers thatallowed the detection of their genetically-restricted lytic activitywithout in vitro restimulation.

TSA-1 plasmid DNA-based vaccine significantly protects mice from T.cruzi-induced mortality. Having established that B6 and BALB/c micegenerated T. cruzi-specific immune responses upon immunization witheither of the TSA-1-expressing constructs, we next determined whetherDNA vaccination could provide these animals with any degree ofprotection against challenge with T. cruzi. Two weeks after the secondimmunizing dose, groups of B6 and BALB/c mice were challenged with 10⁵or 10³ T. cruzi BFT, respectively. The differences in the challengingdose was to compensate for the observed differences in susceptibility ofeach strain of mice. Both strains of mice showed a significant degree ofprotection against T. cruzi-induced mortality. As illustrated in one of3 conducted experiments, B6 mice vaccinated with either of theTSA-1-encoding vectors showed a 7-day delay in the onset of parasitemiaand a consistently reduced level of parasites compared to controlanimals immunized with the unmodified VR1012 vector (FIG. 4A). Moreover,all control animals died before 45 days post-infection, whereas 50% ofmice in each of the test groups survived the infection (FIG. 4B). In thecase of BALB/c mice, however, the steady increase in parasitemia notedin TSA-1 DNA vaccinated animals was strikingly similar to the kineticsof infection observed for mice immunized with the unmodified plasmid DNA(FIG. 5A). Despite similar levels of circulating parasites in test andcontrol animals, none of the mice vaccinated with either of theTSA-1-encoding vectors succumbed to T. cruzi infection whereas 75% ofcontrol mice developed fatal infections within 27 days post-infection(FIG. 5B). Overall, protection against and otherwise lethal innoculumwith trypomastigotes was observed in 73% and 55% of VR1012 TSA1.7- andVR1012 TSA2.1-vaccinated B6 mice, respectively, and in 91% and 86% ofsimilarly vaccinated BALB/c mice (Table 1). In contrast, controlVR1012-vaccinated remained highly susceptible to T. cruzi-inducedlethality as only 9% overall survival was observed for both strains(Table 1).

TABLE 1 Protection against lethal T. cruzi challenge conferred by DNAvaccination B6 mice BALB/c mice Survivors/ Percent Survivors/ PercentPlasmid DNA Challenged survival Challenged survival VR1012 0/3 0/3 0/41/4 1/4 0/4 Total  1/11 9  1/11 9 VR1012 TSA1.7 3/3 3/3 2/4 4/4 3/4 3/4Total  8/11 73 10/11 91 VR1012 TSA2.1 2/3 2/3 2/4 4/4 2/4 nd Total  6/1155 6/7 86 nd: not determined

Discussion

Thus far, the exploration of vaccines against T. cruzi has been widelyavoided due to the fear that such intervention methods would exacerbaterather than prevent a disease that many still consider to have anautoimmune etiology. However, we believe that T. cruzi persists in thediseased tissue, and that it is the persistence of the parasite and notthe parasite-induced immune responses to self molecules which correlatesbest with the induction and maintenance of the inflammatory diseaseprocess. This link between parasite load and severity of disease isfurther supported by the very important role that CD8⁺ T cells play inparasite control and survival to infection. CD8⁺ T cells constitute themajor component in inflammatory foci of T. cruzi-infected tissues and,in their absence, infected mice have increased mortality rates andtissue parasite loads with a decreased or absent inflammatory response.The recent demonstration of CD8⁺CTL in T. cruzi-infected mice and humanswith a specificity for defined trypomastigote and amastigote surfacemolecules and of the immunoprotective phenotype that these cells expressprompted us to initiate the development of immunization strategies tofurther characterize the vaccine potential of parasite components knownto be targets of protective anti-T. cruzi immune responses.

DNA-based immunization has been shown in animal models to easily, safelyand effectively elicit and modulate the spectrum of immune responsesnecessary for the prevention of infectious diseases (S. Gurunathan etal., J. Exp. Med. 186:1137-1147 (1997); D. Lowrie et al., Vaccine15:834-838 (1997): M. Sedegah et al., Proc. Nat'l Acad. Sci. U.S.A.91:9866-70 (1994); J. Ulmer et al., Science 259:1745-1749 (1993); Z.Xiang et al., Virology 199:132-140 (1994); M. Yokoyama et al., J. Virol.69:2684-2688 (1995)) and for the treatment of neoplastic (R. Corny etal., Semin Oncol. 23:135-147 (1996); K. Irvine et al., J. Immunol.156:238-245 (1996)); R. Schirmbeck et al., J. Immunol. 157:3550-3558(1996)), allergic ENRfu (C. Hsu et al., Nat. Med. 2:540-544 (1996); E.Raz et al., Proc. Nat'l. Acad. Sci. USA. 93:5141-45 (1996)) andautoimmune disorders (A. Waisman et al., Nat. Med. 2:899-905 (1996)).Thus, we chose this vaccination method to induce T. cruzi-specificantibody and class I-restricted CD8⁺CTL responses in two inbred mousestrains and to assess its protective efficacy against parasitechallenge. Our recent demonstration of TSA-1 as a target of protectiveCTL made this parasite molecule a prime model antigen to evaluate thisimmunization method inasmuch as the N-proximal portion of TSA-1 hadalready been shown to induce antibody responses which correlate withsurvival to lethal T. cruzi infection and TSA-1 is a member of the large85-kDa family of trypomastigote surface proteins which are recognized byhuman sera and rodent-derived protective antibodies.

Plasmid DNA vaccines VR1012 TSA1.7 and VR1012 TSA2.1 were constructed todrive the expression of products TSA-1₇₈₋₆₅₂ and TSA-1₇₈₋₇₉₀ truncatedat the N-terminus by 77 residues and at the C-terminus by 183 and 45amino acids, respectively. The main reasons for such a design weretwofold: first, because removal of the N-terminal endoplasmic reticulumtranslocation signal sequence would ensure the cytoplasmic retention ofde novo synthesized TSA-1 protein, their subsequent cytosolicdegradation and an efficient priming of CTL responses; second, becauseconventional TSA-1 protein-based immunization of BALB/c mice has shownthat the C-proximal portion encompassing residues 618 to 835 containsepitopes which interfere with the generation of antibodies to theprotective determinants within residues 78 to 619 of the N-proximalportion.

Both VR1012 TSA-1 constructs directed the in vitro expression ofcytoplasmically-retained products with immunoreactivity to sera from T.cruzi-infected mice and in BALB/c mice, both TSA-1-encoding vectors,with and without repeat sequence, elicited parasite-specific antibodyresponses. Such responses were detected after the priming dose, and amodest boosting was achieved after the second dose with the VR1012TSA2.1 vector. In B6 mice, parasite-specific antibodies were detectedonly after the second dose of the VR1012 TSA2.1 vector alone.

The fact that immunization with TSA-1-expressing plasmid DNA vaccinesefficiently elicited MHC class I-restricted CTL responses in B6(H-2^(b)) and BALB/c (H-2^(d)) mice is notable, inasmuch as prior tothese studies T. cruzi-specific CD8⁺CTL had only been primed by parasiteinfection and TSA-1 had only been identified as a CTL target molecule ofB6 mice. The demonstration in B6 mice that TSA-1 DNA vaccination and T.cruzi infection were able to prime CD8⁺CTL populations with specificityfor the same protective H-2K^(b)-restricted ISA-1₅₁₅₋₅₂₂ epitopeindicated that similar immunogenic peptides are generated when a cell istransiently transfected in vivo or when it is expressed by an infectedcell. In agreement with other studies where DNA immunization has beenfound to elicit long-lasting CTL responses, ISA-1₅₁₅₋₅₂₂-specific CTLwere still detected 7 months after administering the last dose of theTSA-1-encoding DNA. The longevity of the response may be explained bythe persistence of the plasmid vaccine in vivo, or to recent reportswhich indicate that CTL memory does not require antigen persistence orCD4 T cell help. Regardless of the mechanisms involved, the ability ofgenetic immunization to maintain a long-lasting response to protectiveT. cruzi CTL epitopes may have significant potential for the developmentof DNA vaccines capable of preventing or treating an established T.cruzi infection.

While the presence of class I-restricted CTL to T. cruzi-infected cellshas been demonstrated in BALB/c mice, the identification of their targetantigens has not been. Hence, in the absence of known TSA-1-derivedH-2^(d)-restricted CTL peptide epitopes, two alternative strategies wereused to determine that TSA-1-expressing DNA vaccines had successfullyprimed parasite antigen specific CTL responses. In the first strategywhere the CTL assay was performed on immune SC without in vitrostimulation significant genetically restricted CTL reactivity wasdetected against T. cruzi-infected target cells. These results suggestthe priming of a substantial number of TSA-1-specific CTL precursors ofwhich a large population remain in a state of activation that allows fortheir direct detection two weeks after the last dose of the DNA vaccine.Similar findings on the detection of CTL activity using unstimulated SCfrom mice immunized with DNA vaccines have been reported for the Vif andNef proteins of HIV-1 and for the SV40 T antigen. In the secondstrategy, the stimulating and targeting activities of T. cruzi-infectedcells were used to confirm the specificity and MHC class I-restrictedlytic activity displayed by in vitro expanded CTL precursors. Thesefindings and the fact that the lytic activity was CD8⁺ T cell-dependentindicate that the observed response was T cell- and not NK cell-mediatedand attest to the value of this method of immunization for primingpotent MHC class I-restricted CTL responses in vivo.

Perhaps the most significant finding of these studies was thatvaccination with TSA-1-expressing plasmid DNA afforded B6 and BALB/cmice significant levels of protection against lethal T. cruzi challengeinfection. Overall survival rates in B6 mice vaccinated with VR1012TSA1.7 or VR1012 TSA2.1 were 73 and 55%, respectively. The sameconstructs furnished BALB/c mice with nearly complete protection as 91and 86% of vaccinated animals survived T. cruzi infection. These resultsare in sharp contrast to the 9% survival observed in animals immunizedwith the unmodified VR1012 plasmid for both strains of mice. It shouldbe noted, though, that immunization with the TSA-1-encoding vectors didnot prevent recipient mice from getting infected, and DNA-vaccinatedmice from both strains developed parasitemias, albeit at differentlevels. In B6 mice, the number of circulating parasites in test animalswas lower than that observed for recipients of the control DNA vaccine,whereas in BALB/c mice, parasitemias were frequently similar in bothgroups of animals.

Example II Genetic Immunization Elicits Antigen-Specific ProtectiveImmune Responses and Decreases Disease Severity in Trypanosoma CruziInfection

Mice and parasites. Six- to 8 week old female C57BL/6J (“B6”) (JacksonLaboratory, Bar Harbor, Me.) were used in all experiments. The Brazilstrain of T. cruzi was maintained in vivo by serial biweekly passage of10³ blood-form trypomastigotes (BFT) in C3H/HeSnJ mice and by continuousin vitro passages of tissue culture-derived trypomastigotes inmonolayers of Vero cells, according to standard parasitology techniques.

Cell lines and culture reagents. Vero (African Green monkey kidneycells, ATCC CCL 81, Rockville, Md.) and RMA-S cells (an immuno-selectedvariant of the RBL-5 lymphoma that is deficient in the expression ofclass I MHC molecules due to a mutation in the TAP-2 peptidetransporter, a gift from Dr. M. B. Oldstone, Scripps Research Institute,La Jolla, Calif.) were maintained in complete RPMI-1640 medium(Mediatech, Herndon, Va.) containing 10% heat-inactivated fetal bovineserum (HyClone, Logan, Utah), 20 mM HEPES, 2 mM L-glutamine, 1 mM sodiumpyruvate, and 50 μg/ml gentamicin (all from Gibco BRL, Gaithersburg,Md.). COST cells (SV40 transformed African Green monkey kidney cells;ATCC CRL 1651) were grown in similarly supplemented Dulbecco's modifiedEagle's medium (DMEM) (Mediatech, Herndon, Va.). T cell medium (TCM) wasprepared by supplementing RPMI-10% FBS with 50 μM 2-mercaptoethanol and0.1 mM non-essential amino acids (Gibco BRL).

Peptides. Peptides were synthesized using Fmoc-based solid phasechemistry on an ACT MPS 350 peptide synthesizer (Advanced Chem Tech,Louisville, Ky.) by the Molecular and Genetic Instrumentation Facility(MGIF) at the University of Georgia. The synthetic peptides pep77.2(TSA-1₅₁₅₋₅₂₂; VDYNFTIV (SEQ ID NO:6)) (B. Wizel et al., J. Immunol.159:6120-30 (1997); M. Santos et al., Mol. Biochem. Parasitol. 86:1-11(1997)), PA8 (ASP-2₅₅₂₋₅₅₉; VNHRFTLV (SEQ ID NO:7)) and PA14(ASP-1₅₀₉₋₅₁₆; VNHDFTVV (SEQ ID NO:8)) (H. Low et al., J. Immunol.160:1817-1823 (1998)) represent H-2K^(b) restricted CTL epitopes from T.cruzi proteins TSA-1, ASP-2 and ASP-1, respectively. TheH-2K^(b)-restricted chicken ovalbumin CTL epitope OVA₂₅₇₋₂₆₄ (SIINFEKL;SEQ ID NO:9) was used as a control peptide (0. Rotzschke et al., Eur. J.Immunol. 21:2891-4 (1991)). Lyophilized peptides were dissolved at 5 mMconcentration in sterile phosphate buffer saline (PBS, 10 mM Na₂HPO₄, 2mM KH₂PO₄, 137 mM NaCl, 8 mM KCl, pH 7.4) and stored at −20° C.

Plasmid construction. The cDNA fragment of TSA-1, ASP-1 and ASP-2 genes(D. Fouts et al., Mol Biochem Parasitol. 46:189-200 (1991); M. Santos etal., Mol. Biochem. Parasitol. 86:1-11 (1997); H. Low et al., Mol.Biochem. Parasitol. 88:137-49 (1997)) encoding amino acid residues78-652, 27-641, 61-705, respectively (excluding the N-terminalendoplasmic reticulum (ER) targeting signal sequence and the C-terminalGPI-anchor cleavage/attachment site and hydrophobic tail), wereamplified by PCR. The recombinant pBluescript II SK⁺ vectors containingTSA-1 (a gift from Dr. David Fouts, University of California, Irvine,Calif.), ASP-1 and ASP-2 were used as template for PCR reactions.Forward and reverse oligonucleotides for amplification of TSA-1, ASP-1and ASP-2 cDNA were designed to incorporate, respectively, BamHI andHindIII, BglII and XhoI, and BglII and SmaI restriction sites(underlined below) for directional cloning. Oligonucleotides wereconstructed on an Applied Biosystems 394 DNA/RNA synthesizer (FosterCity, Calif.) at the MGIF. The forward and reverse oligonucleotides usedfor PCR amplification were 5′-AGGATCCATGATTGCATTTGTCGAAGGC-3′ (SEQ IDNO:10) and 5′-AAAGCTTCATAGTTCACCGACACTCAGTGG-3′ (SEQ ID NO:11) forTSA-1; 5′-AAGATCTTGTGGAAAGGAATTTGAGG-3′ (SEQ ID NO:12) and5′-ACTCGAGTCACAGTGGGCGGTTGTACAG-3′ (SEQ ID NO:13) for ASP-1; and5′-AAGATCTCTGTGAGGCTGCAGACGCTG-3′ (SEQ ID NO:14) and5′-ACCCGGGTTATTGGTCGCCACCGTTTCC-3 (SEQ ID NO:15) for ASP-2. Theamplification products containing the A overhangs generated by Taq DNApolymerase during the PCR reaction were cloned in pUC19(T) vector.

For expression in mammalian cells, the inserts from recombinant pUC19(T)plasmids were excised and cloned in the pCMVI.UBF3/2 plasmid (providedby Drs. Kathryn Sykes and Stephan A. Johnston, University of TexasSouthwestern Medical Center, TX) (FIG. 6). To constructpCMVI.UBF3/2.TSA-1, pUC19(T)TSA-1 was digested with BamHI and HindIII,and the 1.7 kb TSA-1 fragment was cloned in pCMVI.UBF3/2 at similarrestriction sites. To construct pCMVI.UBF3/2.ASP-1, pUC19(T)ASP-1 wasdigested with BglII and Mini, and the 1.8 kb ASP-1 fragment was clonedin pCMVI.UBF3/2 at BglII and SalI sites. pCMVI.UBF3/2.ASP-2 wasconstructed by cloning at BglII and SmaI sites the 1.8 kb ASP-2 fragmentderived from pUC19(T)ASP-2 after digestion with similar restrictionenzymes. The eukaryotic expression vectors encoding murine cytokineIL-12 (pcDNA3.msp35 and pcDNA3.msp40) and murine GM-CSF (pCMVI.GM-CSF)were provided by Dr. S. A. Johnston. Recombinant plasmids weretransformed into E. coli DH5-alpha competent cells, grown in L-brothcontaining 100 μg/ml ampicillin, and purified by anion exchangechromatography using the Qiagen maxi prep kit (Qiagen, Chatsworth,Calif.) according to manufacturer's specifications.

Gene expression. Expression of the ASP-1, ASP-2 and TSA-1 was assessedby transient transfection of COS7 cells with pCMVI.UBF3/2.ASP-1,pCMVI.UBF3/2.ASP-2 and pCMVI.UBF3/2.TSA-1, respectively substantially asdescribed in Example I. Briefly, COS7 cells seeded in 6-well plates(1×10⁵ cells/well) were transfected with 5 μg of each plasmid DNA usingLipofectin (Gibco BRL). After 48 hours of incubation, cells weretransferred to 8-well Lab Tek chamber slides (Nunc Inc., Naperville,Ill.) at 1×10⁴ cells/well, and incubated for an additional 24 hours.Cells were fixed with ice cold methanol, and blocked with 1% bovineserum albumin (BSA) in PBS. After blocking, COS7 cells transfected withpCMVI.UBF3/2.TSA-1 or pCMVI.UBF3/2.ASP-1 were incubated with polyclonalanti-T. cruzi serum (1:200 dilution in PBS-1% BSA) obtained from acutelyor chronically infected mice, respectively. Cells transfected withpCMVI.UBF3/2.ASP-2 were incubated with rabbit anti-ASP-2 polyclonalantiserum (1:200 dilution in PBS-1% BSA). Cells incubated with normalmouse or rabbit serum were used as negative controls. Cells were thenstained with fluorescence isothiocyanate (FITC)-labeled F(ab′)₂ goatanti-mouse or anti-rabbit IgG (1:50 dilution in PBS-1% BSA, SouthernBiotech., Birmingham, Ala.) (10). Slides were mounted in 10% glycerol,0.1 M sodium bicarbonate (pH 9), 2.5% 1,4-diazobicyclo-(2,2,2) octane,and visualized by laser scanning confocal microscopy (MRC-600, Bio-RadLaboratories, Hercules, Calif.).

DNA immunization and infection. Female C57BL/6 mice (6 per group) wereinjected in the quadriceps muscles either with individual plasmid(pCMVI.UBF3/2.ASP-1, pCMVI.UBF3/2.ASP-2 or pCMVI.UBF3/2.TSA-1, 100 μgDNA/mouse) or with a mixture of ASP-1, ASP-2 and TSA-1-encoding vectors(33 μg of each plasmid/mouse). In some experiments, an additional 100 ofcytokine encoding DNA (pcDNA3.msp35, pcDNA3.msp40 [IL-12] andpCMVI.GM-CSF, 33 μg each) was injected along with the antigen-encodingDNA. Mice were boosted 6 weeks after the primary immunization with anidentical dose of plasmid DNA. Two weeks after the second immunization,mice were infected by intra-peritoneal injection of lethal dose of T.cruzi blood-form trypomastigotes (10⁵/mouse, 5 mice per group).Parasitemia were monitored using hemacytometer counts of 10 μl tail-veinblood. Mortality was recorded daily.

Measurement of antibody responses. Cell lysates obtained fromculture-derived T. cruzi (70% amastigotes and 30% trypomastigotes,1.0×10⁹ parasites/ml) was used as a source of T. cruzi soluble antigensfor capturing serum antibodies. Pooled serum samples from immunized micecollected 2 weeks after the first and second immunization were stored at−20° C. until assayed for anti-T. cruzi antibodies by enzyme-linkedImmunosorbant assay (ELISA) substantially as described in Example I.Briefly, flexible U bottom (96 well) polyvinyl chloride plates (BectonDickinson & Co., Oxnard, Calif.) were coated overnight at 4° C. with 100μl/well of T. cruzi soluble antigen (5×10⁵ parasite equivalent/well).Plates were blocked for 2 h at 37° C. with 200 μl/well of 1% non-fat drymilk (NFDM) in PBS. After washing with PBS-0.05% Tween-20 (PBST) andPBS, plates were incubated for 2 hours with test sera (100 μl/well)added in two-fold dilutions in triplicate. Plates were then incubated atroom temperature for 30 minutes with 100 μl/well of horseradishperoxidase-labeled goat anti-mouse immunoglobulin (IgG+M, 1:2000dilution in PBST-1% NFDM, Cappel, West Chester, Pa.). Color wasdeveloped with 100 μl/well of ABTS(2,2′-azino-di-(3-ethyl-benzthiazoline sulfonate) and the opticaldensity was read at 405 nm using an automated ELISA microplate reader(Bio-Tek Instruments, Winooski, Vt.).

To determine the sub-class of antibodies generated in response togenetic immunization, plates coated with T. cruzi lysate were incubatedfirst with test:sera (1:200) for 2 hours at 37° C. and then withbiotin-labeled goat anti-mouse-IgM, -IgG2a, -IgG2b or -IgG1 (1:1000dilution in PBST-1% NFDM, Southern Biotech. Birmingham, Ala.) for 1 hourat 37° C. After washing with PBS-T and PBS, plates were incubated withstreptavidin-conjugated horseradish peroxidase (1:2000 dilution inPBS-T, Cappel, Cochranville, Pa.). Color was developed and measured asabove. Standard deviation was derived from an average of 3 replicates.

Measurement of interferon-γ (IFN-γ). Serum samples pooled from 6 mice ineach group were obtained 2 weeks after the second immunization andanalyzed for the IFN-γ level by specific ELISA substantially asdescribed in G. Nabors et al. (J. Immunol. 146:3591-8 (1991)). Briefly,96 well polystyrene plates were coated overnight at 4° C. with 100 μl ofrat anti-mouse IFN-g Ab (R4-6A2, 4 μg/ml in PBS). After blocking for 2hours at 37° C. with PBST-1% BSA, serum samples were added in triplicatein several two fold dilutions and incubated for 2 hours at 37° C. Fordetection of the IFN-γ protein, plates were incubated for 1 hour withrabbit anti-mouse IFN-γ antibody (1:1000 dilution in PBST-1% BSA). AfterIncubation with horseradish peroxidase-labeled goat anti-rabbit IgG(1:1000 dilution in PBST-1% BSA, Southern Biotech.), color was developedas above. Recombinant mouse IFN-γ was used to generate a standard curve.

Cytotoxic T cell activity. Cytotoxic T cell activity (CTL) activity ofeffector T cells obtained from plasmid DNA immunized C57BL/6 mice wasmeasured 2 weeks after the second immunization. Effector cells weregenerated by incubating 5×10⁶ immune spleen cells (2.5×10⁶ cells/ml TCM,2 ml/well in 24 well plates) with 1 μM immunogenic peptide. Following 2days of culture at 37° C. in 6% CO₂, the culture medium was supplementedwith 5% Rat T-STIM without Con A (Collaborative Biomedical Products,Bedford, Mass.) and incubated for 4 additional days. For targets,RMA-S(H-2K^(b)) cells pre-incubated for 24 h at 29° C., 6% CO₂ wereseeded into 24-well plates (Costar, Cambridge, Mass.) at 10⁶ cells/wellin 2 ml RPMI-10% FBS. Cells were incubated overnight at 37° C. in thepresence of 100 μCi of Na₂ ⁵¹CrO₄ (Amersham, Arlington Heights, Ill.)and 1 μM of PA14 (ASP-1₅₀₉₋₅₁₆), PA8 (ASP-2₅₅₂₋₅₅₉) or pep77.2(TSA-1₅₁₅₋₅₂₂) peptide, or OVA₂₅₇₋₂₆₄ negative control peptide.Cytolytic activity of effectors against targets was measured by the ⁵¹Crrelease assay, as previously described (H. Low et al., J. Immunol.160:1817-1823 (1998); B. Wizel et al., J. Clin. Invest. 102:1062-71(1998)).

Treatment of mice with neutralizing antibodies. In some experiments,mice immunized with genetic vaccines were depleted of specific T cellpopulations by treatment with anti-CD8 (H35.17.2, a gift from Dr.Richard Titus, Colorado State University, Fort Collins) or anti-CD4(GK1.5, American type culture collection, Rockville, Md.) antibodiessubstantially as described in R. Tarleton (J. Immunol. 144:717 (1990)).Antibody treatment of mice started the day of infection and the specificdepletion of lymphocytes was confirmed by flow cytometric analysis ofsplenocytes.

Histology. Some mice were sacrificed during the acute (30-45 dayspost-infection) or chronic (85-240 days post-infection) phase of T.cruzi infection for histological examination of heart and skeletalmuscle tissue. Heart and skeletal muscle tissue was removed and fixed in10% buffered formalin for 24 hours, dehydrated in absolute ethanol,cleared in xylene and embedded in paraffin. Sections (5 μm) were stainedwith hematoxylin and eosin and evaluated by light microscopy. Tissueparasite burden was quantitated based upon the number of parasiticpseudocysts present in sections of heart and skeletal muscles obtainedfrom immunized/infected mice. Tissue sections were screened in >50microscopic fields (mfs) to assess the parasite load. Tissues were alsoscored according to extent of inflammation.

Results

In vitro expression of T. cruzi proteins. T. cruzi genes encoding ASP-1(amino acids 27-641), ASP-2 (amino acids 61-705), and TSA-1 (amino acids78-652) were cloned in CMVI.UBF3/2 mammalian expression vectorcontaining the cytomegalovirus (CMV) immediate-early gene promoter, asynthetic intron and a modified 3′ UR. (untranslated region) from thehuman growth hormone (HGH) (FIG. 6). The cloned genes were fused to aubiquitin encoding gene at the 5′ end (FIG. 6) to promote targeting ofthe expressed protein to the proteosome and entry into the MHC class Ipathway of antigen presentation. The expression of ASP-1, ASP-2 andTSA-1 was determined by antibody staining of COS7 cells transientlytransfected with CMVI.UBF3/2.ASP-1, CMVIUBF3/2.ASP-2 orCMVIUBF3/2.TSA-1, respectively. The intense immunofluorescent stainingwith a polyclonal anti-T. cruzi serum of COS7 cells transientlytransfected with CMVIUBF3/2.ASP-1 or CMVIUBF3/2.TSA-1 confirmed thecytoplasmic expression of ASP-1 and TSA-1, respectively. The cytoplasmicexpression of ASP-2 in CMVIUBF3/2-ASP-2 transfected COS7 cells wasconfirmed by immunofluorescent staining with a rabbit anti-ASP-2polyclonal antiserum. In contrast, no fluorescence was detected whentransfected COS7 cells were stained with normal mouse or rabbit serum,nor using either sera in cells transfected with the vector DNA alone.

Induction of T. cruzi specific humoral and cellular immune responses byDNA immunization. The induction of T. cruzi-specific antibodiesfollowing intramuscular immunization of mice with DNA vaccines wasdetermined by ELISA. T. cruzi-specific antibodies were not detectable insera collected after the first immunization. The presence of parasitespecific antibodies in sera of immunized mice was assessed two weeksafter the second immunization by ELISA. Sera from normal mice (NMS) andmice chronically infected with T. cruzi (CMS) were used as negative andpositive controls, respectively. Moderate levels of parasite-specificantibodies were detected in the sera of mice immunized withCMVIUBF3/2.ASP-2 (FIG. 7A), but remained below the limit of detection insera from mice immunized with CMVIUBF3/2.ASP-1 or CMVIUBF3/2.TSA-1.Likewise, control mice immunized with vector DNA alone or cytokinevectors only exhibited no parasite-specific antibodies in the sera.

ASP-1, ASP-2 and TSA-1, all contain H-2K^(b)-restricted epitopes thatare recognized by CTLs induced in T. cruzi infected C57BL/6 mice (B.Wizel et al., J. Immunol. 159:6120-30 (1997); H. Low et al., J. Immunol.160:1817-1823 (1998)). We determined whether antigen-specific CTLs wereinduced in mice following immunization with the ASP-1, ASP-2 or TSA-1encoding plasmid DNA constructs. Two weeks after second immunization,splenocytes from one mouse in each group were harvested and stimulatedin vitro with ASP-1, ASP-2 or TSA-1 derived H-2K^(b) restricted CTLepitope peptides (PA14, PA8 and pep77.2 respectively, 1 μM). Theeffectors derived from these cultures were tested for their ability tolyse ⁵¹Cr labeled RMA-S cells pulsed with homologous peptide(s) or theirrelevant peptide OVA₂₅₇₋₂₆₄. Moderate levels of peptide-specific lyticactivity were evident in mice immunized with CMVI.UBF3/2.ASP-1,CMVI.UBF3/2.ASP-2 or CMVI.UBF3/2.TSA-1 (FIG. 8A). Splenocytes from miceimmunized with vector DNA did not show CTL activity against any of thepeptide-sensitized target cells (FIG. 8A).

Enhancement of T. cruzi specific immune responses by co-delivery ofcytokine adjuvants. IL-12 and GM-CSF have shown utility as geneticadjuvants, and in addition have been shown to be very important incontrolling the disease outcome to T. cruzi infection (E. Olivares Fonttet al., Parasite Immunol, 17:135-41 (1995); E. Olivares Fontt et al.,Infect. Immun. 64:3429-34 (1996); I. Abrahamsohn, Braz. J. Med. Biol.Res. 31:117-21 (1998); J. Silva et al., Braz. J. Med. Biol. Res.31:111-5 (1998)). We determined whether the immune response to T. cruzigenetic vaccines is enhanced by co-administration of DNA expressionconstructs encoding IL-12 and GM-CSF. Immunization of mice with cytokineadjuvants and antigen-encoding plasmid(s) resulted in splenomegaly, anindicator of increased B and T cell proliferation. In addition, asignificant increase in the level of parasite specific humoral andcellular immune responses was observed in mice immunized with cytokineadjuvants and the antigen-encoding vector(s) relative to mice immunizedwith antigen-encoding vector alone (FIGS. 7 and 8).

The antibody levels in sera of mice injected with cytokine-expressionconstructs and CMVI.UBF3/2.ASP-1 or CMVI.UBF3/2.ASP-2 were comparable tothat of mice chronically infected with T. cruzi (FIG. 7A). Miceimmunized with pCMVI.UBF3/2.TSA-1 and cytokine-encoding plasmids alsoexhibited a significant level of serum antibodies (FIG. 7A). Mouse serapooled from individual tail blood samples two weeks after the secondimmunization were tested for the presence of parasite specific antibodysub-types by ELISA. The increase in antibody response in mice immunizedwith cytokine-encoding plasmids and CMVI.UBF3/2.ASP-1, CMVI.UBF3/2.ASP-2or CMVI.UBF3/2.TSA-1 was evident in IgG1, IgG2a and IgG2b antibodysubclass (FIG. 7B).

Delivery of cytokine-encoding vectors with the antigen-encoding plasmidssignificantly enhanced the activation of antigen-specific CTLs (compareFIGS. 8A and B). Splenocytes obtained 2 weeks after second immunizationwere stimulated in vitro with antigen-specific peptides. In comparisonto splenocytes obtained from mice immunized with CMVI.UBF3/2.ASP-1,CMVI.UBF3/2.ASP-2 or CMVI.UBF3/2.TSA-1, spleen cells from miceco-immunized with cytokine adjuvants and antigen-encoding vector(s)exhibited a 2-4 fold increase in antigen-specific cytolytic activity(FIGS. 8A & B, 40:1 E:T ratio). In addition, IFN-γ in sera pooled fromindividual tail blood samples 2 weeks after the second immunization wasassessed by ELISA. Serum IFN-γ level in mice immunized with CMVI.UBF3/2,CMVI.UBF3/2.ASP-1, CMVI.UBF3/2.TSA-1, or cytokine adjuvants was˜0.05-0.25 ng/ml. Serum levels of IFN-γ were increased by >4-fold whenmice were immunized with cytokine-expressing plasmids plusCMVI.UBF3/2.ASP-1, CMVI.UBF3/2.ASP-2 or CMVI.UBF3/2.TSA-1 as compared tomice immunized with antigen-encoding vector(s) only (FIG. 8C). Theseresults demonstrate that the T. cruzi-specific immune responses elicitedby DNA vaccines can be enhanced by co-administration of cytokineadjuvants.

Protection from lethal T. cruzi infection. We next determined whetherthe immune responses elicited in mice upon immunization with ASP-1,ASP-2 or TSA-1-encoding constructs were protective against T. cruziinfection. Two weeks after the second immunization, mice were challengedwith a lethal dose of T. cruzi (10⁵ BFT/mouse) and blood parasite levelsand mortality was monitored.

All mice injected with ASP-1 DNA vaccine exhibited lower parasitemialevels as compared to either ASP-2 or TSA-1 immunized mice, or controlmice injected with vector DNA (FIG. 9A). As shown in FIG. 9B, 75% ofmice immunized with the CMVI.UBF3/2 encoding ASP-1, ASP-2 or TSA-1survived a minimum of 42 days post-infection (dpi), while 75% miceinjected with the control vector DNA succumbed to T. cruzi infection by42 dpi. In pooled results from 4 experiments, 40% (4/10) of miceimmunized with CMVI.UBF3/2.ASP-1, 66% (10/15) of mice immunized withCMVI.UBF3/2.ASP-2 and 30% (3/10) of mice immunized withCMVI.UBF3/2.TSA-1 were protected from challenge T. cruzi infection while90% (18/20) of mice immunized with CMVI.UBF3/2 succumbed to infection.

The enhanced activation of humoral and cellular immune responses in micereceiving the combination of antigen-encoding plasmids andcytokine-expression constructs was associated with increased resistanceto T. cruzi infection. All mice vaccinated with cytokine-expressingplasmids and CMVI.UBF3/2.ASP-1, CMVI.UBF3/2.ASP-2 or CMVI.UBF3/2.TSA-1efficiently controlled the acute infection; parasitemias becameundetectable 35 days post-infection (FIG. 9C), and all mice survived atleast 70 days post-infection (FIG. 9D). In comparison, all mice injectedwith vector DNA alone exhibited high blood parasite levels and succumbedto T. cruzi infection by 62 days post-infection (FIGS. 9C and D).

Co-immunization with multiple antigen-encoding expression vectorsinduces resistance to T. cruzi infection and Chagas disease. In anattempt to maximize the protective capacity of genetic immunization forT. cruzi infection, C57BL/6 mice were immunized with a mixture of ASP-1,ASP-2 and TSA-1 encoding vectors, with or without cytokine-expressingplasmids. Mice injected with vector DNA or cytokine-encoding plasmidsonly were used as controls. Serum antibody levels were measured usingELISA two weeks after the second immunization. As expected, miceimmunized with antigen (ASP-1+ASP-2+TSA-1)-encoding vectors with orwithout cytokine-expression constructs showed parasite-specific antibodyresponses and CTL activity (FIG. 10). Mice vaccinated with CMVI.UBF3/2encoding ASP-1, ASP-2 and TSA-1 plus cytokine adjuvants (IL-12+GM-CSF)exhibited a 1.5-3-fold increase in parasite-specific serum antibodies(FIG. 10A) and IFN-γ, and an overall increase in antigen-specific CTLactivity in comparison to mice immunized with a mixture ofantigen-encoding constructs alone (FIGS. 10B-E). An inhibitory oradditive effect on elicitation of antigen-specific immune responses wasnot observed in mice immunized with the combination of threeantigen-encoding vectors.

Immunization of mice was followed 2 weeks later by intra-peritonealinfection with blood-form trypomastigotes (10⁵/mouse). Immunization ofmice with multiple antigen-encoding plasmids with or withoutcytokine-expression plasmids induced resistance to T. cruzi infection,although not at a level that was significantly better than that inducedwith single antigen-encoding plasmid (FIG. 11). In pooled results from 2experiments, 58% (7/12) mice immunized with the three antigen-encodingvectors and >80% (10/12) mice immunized with antigen-encoding vectorsplus cytokine-expression plasmids were protected from challenge T. cruziinfection. Interestingly, mice vaccinated with cytokine (IL-12+GM-CSF)adjuvants only exhibited better control of blood parasite burden anddelayed time to death compared to control mice which consistently showedan increase in blood parasite level (FIG. 11A) and succumbed toinfection (FIG. 11B).

Histopathological analysis. The above results suggested that geneticimmunization can be used to generate and modulate the immune responsesthat assure survival from challenge T. cruzi infection. However,survival from T. cruzi infection does not affirm the absence of Chagas'disease. We therefore determined if generation of effector immuneresponses by genetic immunization would result in decreased severity ofdisease during the chronic stage of T. cruzi infection. For thisanalysis, sections from heart and skeletal muscle of mice immunized andchallenged with 10⁵ BFT were assessed at various time intervals fortissue parasite burden and inflammation. Mice were infected two weeksafter second immunization with a lethal dose of T. cruzi BFT(10⁵/mouse). Irrespective of the immunization conditions, mice in allgroups exhibited moderate to high inflammatory responses in skeletalmuscles and heart during acute phase of infection (45 dpi) (FIG. 12,Table 2). However, the extent of tissue parasitism varied depending uponthe immunization conditions. Mice immunized with a mixture ofantigen-encoding vectors plus cytokine-expression constructs exhibitedthe lowest level of tissue parasite burden (0-2 parasite pseudocysts permicroscopic field (mf), FIG. 12D, Table 2).

TABLE 2 Histopathological analysis of the skeletal muscle from DNAimmunized mice upon infection with T. cruzi ^(a) Inflammation^(c) DNAIL-12 + GM- Acute Chronic Pseudocysts/ Immunization CSF^(b) phase^(d)phase^(e) filed^(d) CMVI.UBF3/2 − ++++ +++  8-10 CMVI.UBF3/2 + ++++ +++2-6 ASP-1 − + +++  7-10 ASP-1 + +++ ++ 2-5 ASP-2 − +++ ++ 3-8 ASP-2 +++ + 1-4 TSA-1 − ++/+++ ++ 2-8 TSA-1 + +++/++++   +/++ 2-5 ASP-1 +ASP-2 + − ++++ + 5-9 TSA-1 ASP-1 + ASP-2 + + ++++ +/− 0-2 TSA-1^(a)Results are from a mean of 2-3 experiments. ^(b)When indicated,IL-12 and GM-CSF encoding plasmids (33 μg each/mouse) were administeredwith 100 μg total of antigen-encoding DNA. ^(c)Scores for degree ofinflammation were obtained as described in Materials and Methods.^(d)Tissue were collected for histopathological analysis andquantitation of the parasitic pseudocysis on 45 dpi. ^(e)Tissues werecollected for histopathological analysis during chronic phase ofinfection 80-240 dpi.In comparison, mice injected with ASP-1, ASP-2 or TSA-1-encoding vector(individually) plus cytokine-expression plasmids (1-5 parasite nests/mf)or mice that received the antigen-encoding vector(s) (individually or incombination), cytokine plasmids only, or empty plasmid alone (2-10parasite nests/mf) exhibited moderate to high tissue parasites (FIGS.12A-C, Table 2)

Tissues were collected 150 days post-infection. The extent ofinflammation and associated tissue damage in heart tissue and skeletalmuscle during the chronic phase of infection was remarkably reduced inmice immunized with a mixture of T. cruzi antigen-encoding constructswith or without cytokine-expression constructs (FIG. 13D). Although amajority of the animals in the control group (>90%) died due to highblood and tissue parasite burden during the acute phase of T. cruziinfection, a few animals that did survive to chronic phase of infectionshowed extensive skeletal muscle inflammation and tissue necrosis (FIG.13A, Table 2), the hallmarks of Chagas' disease development. In sharpcontrast, mice surviving infection following immunization with cytokinegenes alone, the mixture of antigen-encoding plasmids, or ASP-1, ASP-2or TSA-1-encoding plasmid plus cytokine-expression vectors; allexhibited moderate inflammatory response up to day 240 post-infection(time point after which experiments were terminated). These resultsdemonstrate that the DNA vaccines used in this study are effective incontrolling the tissue parasite burden and consequently the associatedsymptoms of chronic Chagas' disease.

Depletion of CD4⁺ or CD8⁺T cells and loss of resistance to T. cruziinfection. We next determined whether vaccination with ASP-1, ASP-2 orTSA-1 genes provided protection from T. cruzi infection through CD4⁺and/or CD8⁺T cell dependent immune mechanisms. C57BL/6 female mice wereimmunized twice with vector DNA or a mixture of antigen-encodingplasmids plus cytokine-expressing plasmids, and challenged with1×10⁵/mouse BFT. During the course of infection, some mice (asindicated) were treated with the anti-CD8 or anti-CD4 antibody at weeklyintervals. As determined by flow cytometric analysis, treatment withH35.17.2 or GK1.5 antibodies resulted in specific depletion of >95% CD8⁺or CD4⁺T cells, respectively.

Depletion of either of the T cell population resulted in a decreasedability of immunized mice to control T. cruzi infection. As compared tountreated mice, depletion of either of the CD4⁺ or CD8⁺T cells in micetreated with GK1.5 or H35.17.2 antibodies resulted in a substantialincrease in blood parasite burden (FIG. 14A). All mice in the treatedgroups depleted of either of the T cell population died before day 30 ofinfection. These results suggest that both CD4⁺ and CD8⁺ T cells arerequired for the elicitation of effective immune responses andprotection from T. cruzi infection in DNA immunized mice.

Discussion

In this study, we have determined the utility of T. cruzi genes encodingfor ASP-1, ASP-2 and TSA-1 as genetic vaccine(s) for control of T. cruziinfection and treatment of Chagas disease in a murine model system. Allthree antigens (ASP-1, ASP-2 and TSA-1) are expressed as GPI-anchoredproteins by infective or intracellular stages of T. cruzi, and havepreviously been shown to be recognized by CTLs from chronically infectedmice and humans (H. Low et al., J. Immunol. 160:1817-1823 (1998); B.Wizel et al., J. Clin. Invest. 102:1062-1071 (1998); B. Wizel et al., J.Immunol. 159:6120-30 (1997)). In addition, ASP-1, ASP-2 and TSA-1 elicitstrong antibody responses in T. cruzi infected mice (R. Wrightsman etal., J. Immunol. 153:3148-3154 (1994); M. Santos et al., Mol. Biochem.Parasitol. 86:1-11 (1997); A. Pan et al., J. Immunol. 143:1001 (1989)).

We found that intramuscular immunization of mice with ASP-1, ASP-2 orTSA-1 encoding plasmids (individually or in combination) resulted inelicitation of moderate antigen-specific CTL activity, and marginal tono Ab responses. The restricted induction of antibodies upon delivery ofDNA vaccines via intramuscular route has previously been reported byother labs (M. Barry et al., Vaccine 15:788-91 (1997)). Nevertheless,mice immunized with ASP-1, ASP-2 or TSA-1 encoding vectors exhibited adegree of protection from T. cruzi infection as evidenced by decrease inblood parasite burden and longevity, and control of Chagas' diseaseduring the chronic phase of T. cruzi infection.

To maximize the protective capacity of DNA vaccines to T. cruziinfection, we refined our immunization strategies to enhance themagnitude of the immune responses by co-delivery of immunologicadjuvants along with the antigen-encoding vector(s). IL-12 and GM-CSFwere selected for co-immunization with antigen-encoding vectors. Theimportance of IL-12 and GM-CSF in immune control of T. cruzi has beenshown by the fact that treatment of mice with IL-12 or GM-CSFneutralizing antibodies results in increased susceptibility to T. cruziinfection, while in vivo delivery of rIL-12 or rGM-CSF enhancesresistance to T. cruzi infection. In addition, we and others have shownby infection studies in knock-out mice deficient in various immunefunctions that immunity to T. cruzi requires the induction ofantibodies, CTLs and Th1 type cytokines. Immunization of mice withGM-CSF and IL-12-encoding plasmid DNA along with T. cruziantigen-encoding vectors was expected to elicit more efficient humoraland cellular immune responses and to direct the host immune activitytowards a type1 cytokine response.

Several immunologic effects of co-administration of cytokines with DNAvaccines were observed. First, co-delivery of cytokines resulted insplenomegaly, an indicator of increase in B and/or T cells number andenhanced immune reactivity. In addition, we observed an increase in theinduction of antigen-specific cellular and humoral immune responses andsecretion of Th1 cytokines by co-administration of IL-12 and GM-CSF. Wedetected moderate to no T. cruzi-specific antibodies upon immunizationof mice with T. cruzi gene(s), but a significantly increased antibodyresponse was detected when DNA immunogen(s) were delivered withcytokine-expressing plasmids. The local presence of GM-CSF at the siteof antigen expression might have helped either by increasedproliferation of antigen presenting cells or by increasing theMHC-associated antigen presentation, leading to more effective,antigen-specific B cell activation and antibody production. Enhancementof antibody responses to viral antigens by co-inoculation of DNAencoding GM-CSF has been previously shown (J. Sin et al., Eur. J.Immunol. 28:3530-4038 (1998); J. Kim et al., J. Interferon Cytokine Res.19:77-84 (1999)).

The consistent and dramatic increase in antigen-specific CTL activity inmice co-immunized with cytokine adjuvants and T. cruzi antigen-encodingvectors (individually or in combination) could also be in partattributed to the local presence of IL-12, a key cytokine involved inCD8⁺ T cell activation and proliferation. The lytic activity ofeffectors generated from immunized mice against target cells pulsed withnon-specific peptides was statistically insignificant, suggesting thatthe increase in cytotoxic response by co-administration of cytokines(IL-12) was the result of the activation of antigen-specific Tlymphocytes, not due to non-specific lytic activity of NK cells. As thepeptides used in CTL assays were MHC class I-restricted, we concludethat the CTL activity induced by genetic immunization is MHC classI-restricted and CD8⁺ T cell dependent.

In addition to CTL activity, we also observed an increase in IFN-γsecretion and IgG2a type antibody in mice co-immunized with cytokineadjuvants and antigen-encoding DNA. However, increase in IFN-gproduction in mice receiving cytokine adjuvant was also associated withconsistent increase in IgG1 and IgG2b antibodies. Previous studies haveindicated the increase in IgG1 in immunized mice that exhibit increasedIFN-γ production upon treatment with TL-12 (J. Bliss et al., J. Immunol.156:887-94 (1996)). The co-delivery of IL-12 (and GM-CSF) withantigen-encoding vectors was expected to enhance the type 1 immuneresponse; however, the vaccine cocktail used in the present study wasnot designed to block the production of type 2 cytokines. Therefore thefailure to polarize the T cells completely towards type 1, the inabilityto generate threshold level of IFN-g required for antibody switch toIgG2a, or the production of substantial level of type 2 cytokines (e.g.IL-4) in mice immunized with antigen-encoding plasmid plus cytokineadjuvants might have contributed to the induction of both type 1 andtype 2 antibody subtypes.

Mice immunized with ASP-1, ASP-2 or TSA-1 encoding vectors with orwithout cytokine expressing plasmids exhibited a variable degree ofprotection from T. cruzi infection. The protection was related to theprior induction of immune responses, mice immunized withCMVI.UBF3/2.ASP-2 or the mixture of antigen-encoding vectors pluscytokine adjuvants mounted the maximal immune responses in terms ofanti-T. cruzi antibodies, CTL activity and type 1 cytokines and weremost resistant to T. cruzi infection as evidenced by decreased bloodparasitemia and longevity. The loss of resistance in vaccinated micedepleted of either of the T cell population supports the conclusion thatASP-1, ASP-2 and TSA-1 encoding DNA vaccines conferred protection fromT. cruzi infection through the participation of CD4⁺ and CD8⁺ T cells.In this regard, previous reports from ours and other labs havedemonstrated that both CD4⁺ and CD8⁺ T cells play an important role indetermining the disease outcome in murine model of T. cruzi infection.It is intriguing that mice immunized with cytokine adjuvants were betterequipped to control blood and tissue parasites, and inflammation thanmice injected with vector alone. We consider that the presence of IL-12and GM-CSF at the time of infection (2 weeks following the secondimmunization) might have augmented the parasite-specific responsesinduced by the challenge infection.

The most striking observation in this study is perhaps the demonstrationthat the prior induction of systemic immunity by DNA vaccines reducedthe severity of Chagas disease during chronic phase of T. cruziinfection. The infection experiments in this study were designed suchthat the efficacy of DNA vaccine(s) was determined based upon survivalfrom T. cruzi infection. Therefore, mice were infected with a high doseof BFT (1×10⁵) that was sufficient to kill a majority of the controlanimals during the acute phase of infection, long before the developmentof chronic chagasic symptoms. Under such conditions, mice immunized withantigen-encoding vector(s) and cytokine adjuvants not only survived thechallenge T. cruzi infection, but also controlled the blood and tissueparasite burden and exhibited a dramatic reduction in skeletal and heartmuscle inflammation and necrosis during the chronic phase of T. cruziinfection. It is likely that effective control of parasites early duringthe acute phase of infection resulted in the control of tissue parasiteload and associated Chagas' disease development. Regardless of themechanism, our results provide the first evidence that DNA vaccinationis a viable approach to reduce the severity of chronic T. cruziinfection. Additional experiments using appropriate mouse-T. cruzicombinations so that control animals survive and develop chronic phaseof Chagas' disease are in progress for the comparative analysis and thequantification of effectiveness of DNA vaccines for the prevention ofChagas disease in a variety of models.

In conclusion, we have demonstrated that (i) genetic vaccinesconstituted by ASP-1, ASP-2, TSA-1 are useful for the induction ofimmunity to T. cruzi infection and the prevention of Chagas disease (ii)individual or multiple antigens can be delivered for the elicitation ofantigen-specific immune responses, (iii) the quality and the quantity ofimmune responses elicited by T. cruzi DNA vaccine(s) can be enhanced byco-delivery of cytokine expression plasmids, (iv) the stimulation andmaintenance of antigen-specific CD4 and CD8⁺ T cells is essential forefficient control of T. cruzi infection by genetic immunization.Although the three genes tested in this study failed to prevent T. cruziinfection or eliminate the parasites from infected animals, they didcontrol infection and Chagas' disease.

Example III Therapeutic Genetic Immunization of Mice Infected with T.cruzi Reduces Disease Severity

The protocol and genes used here were the same as with the prophylacticimmunization described in Example II. Mice were infected with 1000blood-faun trypomastigotes of the Brazil strain of T. cruzi and then 4.5months later, immunized with 33 ug each of the TSA-1, ASP-1 and ASP-2plasmids in combination with the IL-12 and GM-CSF-containing plasmids asin Example H. Approximately one year later, these mice were sacrificedand the skeletal and heart muscle subjected to standardhistopathological analysis. Mice which received the empty plasmid orcytokine genes alone showed extensive inflammation and tissue scarringcharacteristic of Chagas' disease. Mice receiving the plasmidscontaining the T. cruzi genes or the combination of T. cruzi genes andcytokine plasmids were essentially free of inflammation and disease(FIG. 15).

Example IV Comparison of Protective Cellular and Humoral ImmuneResponses to Trypanosoma Cruzi Infection in B6 and C3H Mice Using DNAVaccines

Methods. Truncated version of the genes encoding trans-sialidasesuper-family members ASP-1 (amino acids 27-641), ASP-2 (amino acids61-705) and TSA-1 (amino acids 78-652) were cloned in eukaryotic geneticexpression vector CMVI.UBF3/2 as in Example II.

C57BL/6 (B6, H-2^(b)) mice (“B6 mice”) are described in Example II. B6mice (5/group) were immunized twice at an interval of six weeks withantigen (ASP-1, ASP-2 and TSA-1, 1 μg each) encoding vectors with orwithout IL-12 and GM-CSF expression constructs (33 μg each). Two weeksafter the second immunization, mice were challenged with BFT (Brazilstrain, 1×10⁵/mouse) and mortality was recorded daily.

C3H/HeSnJ (C3H, H-2^(k)) mice (“C3H mice”) were obtained from JacksonLaboratories (Bar Harbor, Me.) at 6-8 weeks of age. C3H mice (5/group)were injected with CMVI.UBF3/2 containing ASP-1, ASP-2 and TSA-1 with orwithout cytokines encoding plasmids (33 μg each) as above. The presenceof parasite-specific antibodies was assessed by ELISA using sera pooledfrom individual tail blood samples (5 mice per group) collected 2 weeksafter the second immunization. Negative and positive controls were serafrom normal mice (NMS) and from mice chronically infected with T. cruzi(CMS), respectively. Two weeks after second immunization, animals wereinfected by intraperitoneal injection with 10³ T. cruzi BFT. Bloodparasite levels were monitored using hemacytometer counts of 10 μltail-vein blood.

Multicomponent vaccine in B6 mice. As described in Example 11, B6 miceimmunized with CMVI.UBF3/2 containing ASP-1, ASP-2 and TSA-1 genesexhibited moderate levels of T. cruzi specific antibodies andantigen-specific CTL activity. When challenged with a lethal dose(10⁵/mouse) of T. cruzi trypomastigotes, all mice immunized with ASP-1,ASP-2 and TSA-1 encoding genes exhibited lower parasitemia levels, and60% of the mice survived the challenge infection. Of the mice injectedwith the control vector DNA, 80% succumbed to T. cruzi infection (FIG.16A). However, like immunization with TSA-1 alone (Example I), the threegenes together also failed to provide complete protection from T. cruziinfection. Immunized mice which survived the challenge infectionexhibited varying degrees of inflammation in the heart and/or skeletalmuscles, indicative of the persistent infection.

Multicomponent vaccine with cytokine in B6 mice. IL-12 and GM-CSF haveshown utility as vaccine adjuvants, and in addition have been shown tobe important in controlling disease outcome to T. cruzi infection (E.Fontt et al., Infect Immun. 66:2722-2727 (1998)), C. Meyer zumBuschenfelde et al., Clin. Exp. Immunol. 110:378-385 (1997)). IL-12 andGM-CSF have been reported to enhance both humoral and cellular immuneresponses (Y. Chow et al., J. Immunol. 160:1320-1329 (1998); M. Geissleret al., J. Immunol. 158:1231-1237 (1997); K. Irvine et al., J. Immunol.156:238-245 (1996); J. Kim et al., J. Immunol. 158:816-826 (1997); Z.Xiang et al., Immunity 2:129-135 (1995)). As described in Example II,mice vaccinated with ASP-1, ASP-2 and TSA-1 DNA plus the IL-12 andGM-CSF expression constructs exhibited significantly higher levels of T.cruzi specific antibodies and CTL activity compared to mice immunizedwith antigen encoding expression constructs alone. The enhancement inactivation of both humoral and cellular responses by co-injection ofcytokines encoding vectors correlated with increased resistance to T.cruzi infection, as evidenced by the reduction in blood parasites,tissue parasites and inflammation and increased survival (FIGS. 16A &B).

C3H mice. The ability of genetic vaccines to provide protection from T.cruzi infection in another inbred mouse strain (C3H mice) which is moresusceptible than the B6 mouse strain to infection with Brazil strain ofT. cruzi was examined. Immunization of C3H mice with antigen encodingvectors and cytokine expression plasmids resulted in induction ofsignificant levels of anti-parasite antibodies (FIG. 17A). However, whenchallenged with a lethal dose of BFT (1000/mouse), C3H mice immunizedwith ASP-1+ASP-2+TSA-1 encoding plasmids with or without IL-12+GM-CSFexpression constructs exhibited high parasitemia similar to control miceinjected with vector alone (FIG. 17B). All mice succumbed to infectionirrespective of vaccination by 39 days post-infection (FIG. 17C).

Summary. Following intramuscular immunization with ASP-1, ASP-2 andTSA-1 expression plasmids, C57BL/6 (B6, H-2^(b)) mice exhibited moderatelevel of immunity to T. cruzi infection, which was significantlyenhanced by co-delivery of IL-12 and GM-CSF encoding plasmids. The levelof protection correlated with the amount of antigen encoding vectorsused for immunization. Despite the successful vaccination of B6 mice,immunization of C3H/HeSnJ (C3H, H-2^(k)) mice with plasmid DNAsexpressing ASP-1, ASP-2, TSA-1 with or without IL-12, GM-CSF failed togenerate significant protective immunity. It should be noted, however,that C3H mice are extremely susceptible to infection with T. cruzi(typically 100% mortality is observed upon initial infection) and thuswould have been expected to respond differently. These results dodemonstrate that multicomponent genetic vaccines constituted by ASP-1,ASP-2 and TSA-1 are useful for the induction of partial immunity to T.cruzi infection, and the minimal amount of each DNA required forelicitation of protection from T. cruzi infection is in the range of0.1-1 μg. The quality and the quantity of immune responses can beenhanced by co-delivery of cytokine expression plasmids. The immuneresponse generated by the three genes in B6 mice was significant,although not sufficient to prevent T. cruzi infection or to preventdeath from infection in 100% of vaccinated animals.

Example V Determination of Minimal Plasmid DNA Sufficient to ElicitImmunity

To determine the minimal amount of plasmid DNA sufficient to elicitimmunity to T. cruzi, mice were immunized with various dilutions of eachof CMVI.UBF3/2-ASP-1, -ASP-2 or -TSA-1 genes, (0.001-33 μg/mouse).Co-immunization with up to 10 ng of each gene provided the similarlevels of resistance as provided by 33 μg of each gene, thoughdetectable level of CTL responses were generated only in mice immunizedwith 33 μg of each DNA vaccine. None of these groups of mice exhibiteddetectable antibody responses. When T. cruzi genes were co-deliveredwith cytokine adjuvants, as little as 1 ng of vaccine DNA resulted inactivation of T. cruzi specific, MHC class I restricted T cells capableof cytolytic activity, while 10 ng of each DNA was needed to induce theproduction of moderate levels of antibodies. The degree of reduction intissue and blood parasite load, the intensity of inflammatory infiltratein myocardial tissue and the level of resistance to T. cruzi infectioncorrelated with the amount of DNA delivered, with the maximum protectionbeing observed with 0.1-33 μg of each vaccine DNA and cytokines. All themice immunized with 1-33 μg of DNA along with cytokine adjuvantssurvived a minimum of 90 days post-infection (dpi), in comparison tocontrols which succumbed to infection as early as 35 dpi.

Example VI Identification of Immunogenic Polypeptides Using ExpressionLibrary Immunization (ELI)

ELI is unbiased with respect to antigen type by putting no constraintson nor making any predictions about antigens which should be goodvaccines. ELI is also unbiased with respect to the immune effectormechanism that should be induced to get good protection—DNA vaccinesgenerally elicit both cellular (including CD8⁺-dependent) and humoralresponses and can be designed or delivered to emphasize either or both.

Efficacy of vector pORF.GFP for the selection of ORF-genes. Although wehave already constructed a small library of T. cruzi genomic DNA inpcDNA3, we are in the process of constructing an ORF library of up to50,000 clones in ORF.GFP vector. Screening of the T. cruzi genome forprotective genes by ELI would be greatly facilitated by the generationof an ORF-library. pORF.GFP has been designed by Johnston et al. (TexasSouthwestern Medical Center, TX) for the selection of inserts with longORF in E. coli. An E. coli optimized green fluorescent protein (GFP)encoding gene is inserted in pCMVI, 3′ to the unique restriction site tobe used for cloning of random genomic inserts. A T7 promoter and ATGcodon along with Shine-Delgarno sequence is positioned upstream of BamHIsite, and a T7 termination sequence is provided downstream of GFP. E.coli HMS174 cells which contain T7 polymerase under the control ofisopropyl β-D thiogalactopyranoside (IPTG) inducible LacZ promoter aretransformed with the recombinant plasmids. T₇ polymerase mediatestranscription and translation from the insert-GFP fusion genes if insertDNA is present as a continuous open reading frame in fusion with the GFPencoding gene. Inserts which are out of frame or are oriented in reverseand therefore likely to contain stop codons (considering all codons areutilized at equal frequency, 15 stop codons/kb of non-coding DNA canoccur) would not read through and express GFP. Two versions of ORF.GFPplasmid that will be used for cloning of the T. cruzi genomic DNA areORF.PA.GFP and ORF.PNA.GFP. Both plasmids contain PacI and AscIrestrictions sites surrounding the unique BamH1 (In ORF.PBA.GFP or Nan(In ORF.PNA.GFP) restriction sites for cloning of the random genomicinserts. Genomic DNA digested with Sau3A enzymes can be cloned inpORF.PBA.GFP at the BamH1 site while genomic DNA digested with HpaII,Taq1 or HinPII can be cloned in pORF.PNA.GFP at the NarI.

The fluorescent signal obtained with illumination of GFP expressingbacteria under uv light provides an easy tool for sorting of therecombinants containing ORF-genes. The efficacy of the pORF.PBA.GFPplasmid to provide selection for ORF-genes has been determined byJohnston et al. For this, the bacteria transformed with a yeast or T.cruzi genomic library constructed in pORF.PBA.GFP plasmid were inducedfor GFP expression on LB-agar plates containing 40 μM IPTG and incubatedat 30° C. for 2 days. Recombinants which expressed GFP were manuallypicked by visualization of fluorescent signal obtained upon illuminationwith long wave uv transilluminator. Sequencing of 50 randomly pickedGFP-positive clones from the yeast library and 30 clones from the T.cruzi library suggested the presence of ORF in all of the sequencedclones and unbiased representation of the genome (GFP expression was notbased upon the insertion of specific group of genes or sequences).Sequencing of these clones also confirmed the incorporation of singleinsert/clone and the absence of chimeric fusion genes. Thus, we expectthat selection of bacteria expressing high levels of inducible GFP-basedfluorescence from the library generated with T. cruzi genomic DNA in thepORF.GFP vector will provide a representative ORF-library containingmost T. cruzi genes.

Generation of a T. cruzi ORF-library in a eukaryotic expression vector.The ORF.GFP library clones from each 96-well plate are pooled together.Plasmid DNA isolated from the plate pools is digested with PacI and AscIrestriction enzymes, separated on 1% agarose gels.).2 to 1 kB T. cruzigenomic DNA fragments are purified and ligated in CMV.UB.PBA vectordigested with similar enzymes. The CMV.UB.PBA vector is designed suchthat the T. cruzi genomic DNA inserts are transcribed through strongcytomegalovirus promoter and the translation initiates with a ubiquitinterminal fusion. This vector when used for genetic immunization allowshigh level production of the fusion protein, and the antigenic proteinis targeted to the proteosome for efficient processing and presentation.The T. cruzi genomic library in eukaryotic expression vector CMV.UB.PBAis then screened using the ELI approach for the isolation of individualprotective genes.

Generation of “repeat-less” ORF-library. Although high copy, relatedgene families are in general present in kinetoplastids, the T. cruzigenome is especially rich in repeat gene families. The presence of thesegene families (e.g. trans-sialidases (TS), mucins, cruzipains, L1Tcrepeats etc.) which contain 100s-1000s of members sharing high homologycould be of particular concern when screening the T. cruzi genome forprotective genes. If the majority of members of these gene familiesexhibit protective capability, and are distributed randomly in the96-well plates containing ORF-library, then we may end up identifying TSor other family members only as protective genes during libraryscreening by ELI. We have already demonstrated that a significant levelof protection from T. cruzi infection can be generated by immunizationwith three members (TSA-1, ASP-1, ASP-2) of the TS-gene family(Preliminary Studies). However, 1000s of trans-sialidase family members,which are not known to have any functional significance are consideredto be more susceptible to variation in epitopes and loss among differentstrains of T. cruzi than the house-keeping genes which areevolutionarily more conserved. Therefore, a vaccine constituted ofrepeat genes may not prove to be as effective against different strainsof T. cruzi as a vaccine made of low copy, essential conserved genes.

If the presence of repeat genes inhibits our attempts to identify lowcopy protective genes, we will use a “repeat-less” ORF library (devoidof genes belonging to large gene-families) for screening by ELI. Forthis, ORF-library clones from 96-well plates will be transferred tonitrocellulose filters, uv cross linked, and hybridized at lowstringency with radioactive probes derived from TS family genes (ASP-1,ASP-2 and TSA-1), cruzipain repeats containing conserved sequences,mucin family members containing highly conserved N and C-terminaldomains and L1 Tc repeats. Clones identified as members of the largegene families by dot-blot hybridization then will not be included duringpooling of clones from ORF-library and “repeatless” ORF-library will bescreened by ELI for protective genes.

Evaluation of libraries. The pcDNA3/T. cruzi library clones wereanalyzed for random and complete representation of the T. cruzi genomeby sequencing 30 random clones. Approximately 40% of the sequences areexpected to match the sequences of trypanosomatids or other organismsalready in the public databases. Sequence analysis was performed, andsuggests that this library does not consist of selective gene familymembers only, and that non-family members that are present at low copynumber in T. cruzi genome are represented at the desired frequency. Therandomness of the library will be further confirmed by screening ˜2000randomly chosen clones in the library (20×96-well plates) by dot blothybridization using radio-labeled transialidases, mucins, sires, E1-3repeats and mini/maxi circles probes. The randomness of the library canbe assessed based upon the probability of presence of these genes in T.cruzi genome compared to what is found in the library. To determine thatmajority of the pcDNA3/T. cruzi library clones stored in the librarycontained inserts, 100 clones were analyzed by restriction digestion andPCR amplification. These experiments suggest that ˜70-80% clones in thelibrary contain inserts of various sizes (in the range of 200-500 bp).

The ORF-library will likewise be evaluated for representation of the T.cruzi genome. 5′ and 3′ ends of the inserts from 100 randomly pickedORF-clones will be sequenced. Representation of the genome in theORF-library will also be confirmed by hybridization mapping with ³²Plabeled T. cruzi high copy number genes (genes encoding trans-sialidasefamily proteins, mucins, amastins, tubulins and cruzipain).Hybridization of the ORF-library with low copy number genes (gp72, onecopy, glyceraldehyde phosphate dehydrogenase, 2 copies) will confirm thepresence of a majority of T. cruzi genes in ORF-library. To confirm thatthe genes cloned in pORF.GFP are expressed in mammalian cells, some ofthe randomly picked plasmid preparations from individual and mixtures ofclones will be transiently transfected in COST cells and thetransfectants tested for the expression by immunofluorescent stainingwith polyclonal anti-T. cruzi serum. The lack of GFP expression inmammalian cells will confirm that fusion proteins are not made ineukaryotic cells. This technique has been previously used in our lab todemonstrate the expression of TSA-1, ASP-1 and ASP-2 encoding genescloned in genetic immunization vectors.

Effect of genetic vaccines on protection and survival from T. cruziinfection. The determination of relative efficiency of each sub-library,plate-pool or sets of genes to elicit immune responses and provideprotection from T. cruzi infection is estimated based upon thequantitation of blood and tissue parasites in the acute phase andsurvival from infection.

C57/BL6 mice were immunized twice at an interval of 6 weeks with pcDNA3vector only, or with a plate pool of pcDNA3/T. cruzi library clones (96clones, ˜1 μg each) with/or without IL-12 and GM-CSF cytokine adjuvants(10 μg each). Mice immunized with ASP-1+ASP-2+TSA-1 (1 μgeach)+IL-12+GM-CSF (10 μg each) were used as positive control. Mice werechallenged with 100,000 BFT of T. cruzi (Brazil) two weekspost-immunization, and observed for survival from infection. It wasobserved that 60% of the mice (3 out of 5) immunized with pcDNA3 librarypool with or without cytokine adjuvants, and 80% mice (4 out of 5)immunized with a mixture of ASP-1+ASP-2+TSA-1 (1 μg each)+IL-12+GM-CSFsurvived T. cruzi infection at least 80 days post-infection while allmice injected with control vector (pcDNA3 only) succumbed to T. cruziinfection by 65 days post-infection. These results suggest thatelicitation of protective immune responses by immunization of mice withpools of 96 clones is feasible.

The Brazil strain of T. cruzi is used for infection studies because theinfection and disease development in various mouse strains this strainis well characterized. Screening of SylvioX10/7 ORF-library forprotective genes using Brazil strain for infection will also provideevidence for cross-strain protection. To analyze the ORF.GFP vectorlibrary, BALB/cByJ mice (Jackson laboratories) will be used for thesestudies. These mice exhibit an intermediate resistance to T. cruzi(Brazil strain) depending upon the number of infectious trypomastigotesof the Brazil strain injected. 80-100% mice infected with 5×10⁴−1×10⁵blood-form trypomastigotes succumb to infection within 30-50 dayspost-infection.

Bacterial clones obtained from subcloning of pools of inserts intoCMV.UB.PBA vector will be amplified on LB-agar plates overnight at 37°C. This will assure the growth of slow growing bacterial clones whichare overtaken by fast growing clones when cultured together. Bacterialcultures from each agar plate will then be harvested in a total of 15 mLLB and plasmids purified using EndoFree Plasmid Maxi Kit (Qiagen) andProcipitate (LigoChem Inc). DNA will be injected in the quadricepsmuscle of mice (6/group). Use of endotoxin-free plasmid DNA will ensurethat immune responses are not induced due to injection of bacteriallipopolysaccharides (potent inflammatory agents). Mice will be immunizedtwice at a 45 day interval (we have found this interval to be optimumfor induction of immune responses and protection from T. cruzi infectionin previous studies. Mice injected with the vector alone orCMVI.UBF3/2.ASP-2 will be used as negative and positive controls,respectively.

Two weeks following the second immunization, mice will be challengedwith a lethal dose (10⁵/mouse) of BFT of the Brazil strain of T. cruziby intraperitoneal injection. The efficacy of each pool of genes toprovide protection from T. cruzi infection will be evaluated by thefollowing parameters:

(i) Parasitemia: Parasitemias in tail vein blood will be monitored atweekly intervals by microscopic counting of trypomastigotes present inblood and/or estimating the luciferase activity in transfectant T. cruziin blood using standard techniques. The parasite counting technique istime consuming and is also limiting in sensitivity and accuracy becauseit often misses significant numbers of amastigotes that are present incirculation. Because of these drawbacks, we have recently developed theT. cruzi transfectants expressing fire-fly luciferase and have adaptedmethods for the rapid detection of luciferase activity and thus parasitenumber in blood.

Luciferase expressing Brazil strain transfectants were generated by theintroduction of the firefly luciferase reporter vector pHD421 Bner intoepimastigotes of T. cruzi Brazil strain by electroporation. pHD421Bneris a derivative of the trypanosome expression vector pHD421 in which theT. brucei tubulin sequences were replaced with the T. cruzi beta tubulinsequences to allow targeted integration into the T. cruzi genome byhomologous recombination. Additionally, the native 5′ UR. of theluciferase gene was replaced with the expression enhancer sequencener-ACC (1) at the −1 position. Transfectant epimastigotes were selectedin the presence of hygromycin (1 mg/ml) and luciferase activity isestimated using Promega's Luciferase Assay System and a TropixLuminometer.

(ii) Histological detection of tissue parasites: Although parasitemiaprovides a quick and relatively easy measure of resistance to infection,parasite level is not a good prediction of survival. To obtain a secondmeasure of parasite control, quantitation of the parasite load invarious tissues of immunized/infected mice during the acute phase ofinfection (30-50 days post-infection) will be used to determine theability of genetic immunization to control the infection. For this, onemouse from each group will be sacrificed 30 days post-infection, andheart, liver, kidney, spleen and skeletal muscle tissue will becollected. Tissues will be fixed with 10% formalin, dehydrated inabsolute ethanol, cleared in xylene and embedded in paraffin. Fivemicron thick sections will be cut and stained with hematoxylin & eosinand analyzed using light microscopy. Tissue sections will be examined ina blinded fashion and scored according to extent of tissue parasitism.

(iii) Survival from infection: Mortality will be recorded daily.

We believe that evaluation of infected mice to control the T. cruziinfection based upon the above parameters will provide a relatively easyand quick estimation of the efficacy of genetic vaccines.

Characterization of inserts mediating protection. We are aiming atidentification of 20-50 protective genes by screening of ORF-library byELI. Though full characterization of these genes during the course ofthis project will not be possible, attempts will be made to determinethe function of these genes based upon following parameters:

(i) End sequencing: The genes identified based upon their capability togenerate protection from T. cruzi infection will be sequenced at the 5′and 3′ ends using vector specific oligonucleotide primers. Based uponresults from the sequencing of trypanosomatid genes, we expect to findhomologues for ˜40% of the inserts present in sequenced plasmids. Wewill fully sequence those genes for which no homology is found amongsequences in the public databases. The presence of the N-terminal signalsequence, COOH-terminal cleavage and GPI-anchor addition signalsequence, hydrophobic regions or other motifs will be used to helpdetermine the identification, function and localization of these geneproducts.

(ii) Cloning of complete gene: It is likely that many of the protectiveORF-clones will not contain complete genes. Because we plan to identify20-50 genes, attempts to recover full genes will be done for very few ofthe protective inserts. Those for which full genes might be sought wouldinclude inserts in plasmids that yield very high levels of protectionbut which appear from sequence information to be incomplete and forwhich the available sequence provide no clue as to the identity orfunction of the encoded gene products. Under these situations, we woulduse gene splicing by overlapping extension (SOE) PCR (successfully usedby us to obtain the full sequence of ASP-2 gene) for identification andcloning of the complete gene.

(iii) Functional characterization: Functional analysis will be done foronly a few of highly protective ORF-clones for which complete genes areidentified. To produce the antibodies against these selected geneproducts, mice (3/gene) will be immunized with the genetic vaccine alongwith the GM-CSF encoding plasmid (33 μg each DNA/mouse) twice at aninterval of 45 days. We have successfully used this protocol to produceanti-ASP-I, ASP-2 and TSA-1 antibodies. Staining of the permeabilizedand unpermeabilized parasites (epimastigotes, trypomastigotes andamastigotes) with antibody and visualization by confocal microscopy willdetermine the stage of expression as well as localization of theprotein. Western blotting of the cell lysate from three developmentalstages of T. cruzi with the antibodies will also confirm the stage ofexpression and size of the proteins encoded by those genes. Scanning ofthe microarrays for genes upregulated in different developmental stagesmight provide some information about stage specific expression of someof the protective genes identified by ELI.

Effect of genetic immunization on parasite burden during the chronicphase of T. cruzi infection. To determine if genetic immunizationdecreases the disease severity in chronic stage of infected mice, micewhich survive infection following genetic immunization will be examinedfor tissue parasite load and for the presence of inflammation. Survivingmice at >120 days post-infection will be compared to control miceinjected with empty vector DNA alone, and challenged with 1000trypomastigotes (a majority of the control mice infected with this lowdose of T. cruzi will be able to control the acute infection and surviveinto the chronic phase of chagasic myocardiomyopathy). Mice will besacrificed during chronic phase of T. cruzi infection and analyzed forparasite load and for the severity of disease.

These experiments will assess the effect of immunization on bloodparasite load. However, the level of parasites in the blood does notalways correlate with the tissue parasite load. Also, once the chronicphase of infection is reached, detection of parasites by standardhistological approaches is very difficult. Therefore, the effect ofimmunization on the parasite burden in the heart and skeletal muscles ofmice will be assessed by more sensitive techniques and compared toparasite load in unvaccinated/infected control group mice.

(i) Enzymatic estimation of luciferase activity in various tissues: TheT. cruzi Brazil strain parasites to be used for challenge infection inthese studies is transfected with a gene encoding cytoplasmic luciferase(LUC). These transfectants express a very high level of luciferaseactivity which can be estimated by fairly easy and standard methods(described above). We are already using these transfectants forsensitive and accurate determination of parasite levels as compared tomicroscopy counting in vitro infection studies. To determine the effectof immunization with pools from ORF-library on the parasite burden inheart and skeletal muscles of infected mice, one mouse from eachsurviving group will be sacrificed and proteins from heart tissue andskeletal muscles will be extracted and quantitated for luciferaseactivity using kits supplied by Promega. Comparison of the specificenzyme activity from immunized/infected mice to control infected micewill then determine the effect of immunization in controlling the tissueparasite burden. Number of parasites in a specific tissue will becalculated by reference to a standard curve.

(ii) Competitive PCR: To quantitate the effect of immunization on tissueparasite load, total DNA isolated from various organs of the infectedmice will be used as template for competitive PCR. PCR will be carriedout using T. cruzi kDNA specific oligonucleotides5′-GGTTCGATTGGGGTTGGTGTAATATA-3′ (SEQ ID NO:16) and5′-AAATAATGTACGGGT/GGAGATGCATGA-3′ (SEQ ID NO:17) as forward and reverseprimers respectively to amplify a 330 by fragment. Plasmid pBSKILLcontaining 442 by fragment of IL-2 gene (57-497 bp) as stuffer fragmentflanked by kDNA specific sequences will be used as competitor forperformance of quantitation by competitive PCR.

(iii) Detection of T. cruzi by in situ PCR: In situ PCR amplification ofT. cruzi minicircle kinetoplast DNA (kDNA) has been used to detectparasites or parasite derived DNA in infected tissue. The highsensitivity of this procedure is enhanced by the fact that each parasitehas 5-10,000 copies of minicircles, and each minicircle has 4 copies ofconserved regions, which are to be used as targets for PCR amplificationof kDNA. If parasites can not be detected in immunized mice by enzymaticand competitive PCR techniques, then further confirmation of clearanceof parasites and cure from disease will be done by monitoring thepresence of parasites in tissues of the mice by in situ PCR. Tissuesfrom the heart or skeletal muscle of infected mice will be processed,sectioned and fixed as previously described (60). kDNA-specificoligonucleotides 5′-GGTTCGATTGGGGTTGGTGTAATATA-3′ (SEQ ID NO:18) andbiotin-labeled 5′-AAATAATGTACGGGT/GGAGATGCATGA-3′ (SEQ ID NO:19) asforward and reverse primers will be used for amplification by PCR. PCRproducts will be detected with avidin-peroxidase and color developedwith 3′,3′-diaminobenzidine tetrahydrochloride. Sections will becounter-stained with hematoxylin and visualized by light microscopy. Atleast 200-microscopic fields from different sections of the heart andskeletal muscles tissue of mice will be screened for the presence ofparasites. Comparison of the number of parasitic foci in tissue fromimmunized/infected mice to control infected mice will determine theeffect of immunization in controlling the tissue parasite burden.

Effect of genetic immunization on disease severity. Based upon ourprevious studies, we expect that a reduction in acute phaseparasitization and chronic parasite load will correlate with a reductionin inflammatory disease. To determine the quantitative effect ofimmunization with pools or sets of genes from ORF-library on diseaseseverity in chronically infected mice, techniques well standardized inour laboratory will be used. Heart and skeletal muscle tissue fromimmunized/challenged mice will be fixed, embedded in paraffin, and fivemicron sections stained with hematoxylin and eosin will be analyzedusing light microscopy. Tissue sections from mice infected withoutvaccination will be used as control. Tissue sections will be scored from0 to 4 in blind studies according to the extent of tissue damage fromnormal to total wall involvement.

Example VII Identification of Immunogenic Polypeptides Using DNAMicroarray Technology

DNA microarrays provide the means to monitor the expression of many orall genes in a genome, under different growth conditions, duringdifferent developmental stages, or in different tissues. This technologywill be used to identify genes whose expression is upregulated in T.cruzi during the intracellular amastigote stage of the infectious cycle,soon after infection of the host cell. This focus is based upon thereasoning that the protein products of such genes would be among thefirst to reach the class I MHC processing and presentation pathway andthus would serve as early indicators to the immune system of theinfection status of host cells. We believe that amastigote targetantigens are part of the “missing link” for vaccine development in T.cruzi. We showed in Example II that the CD8⁺ T cell population, theprimary role of which is to recognize and kill host cells harboringintracellular pathogens, is very important for immune control of T.cruzi. Furthermore, we showed that transfer of T. cruzi-specific CD8⁺ Tcell lines, or immunizations which induce parasite-specific CD8⁺ T cellresponses to amastigote-expressed proteins, are protective in murinemodels of T. cruzi infection.

Scanning and arraying equipment. An microarrayer based on the design,plans, and software made available by the Pat Brown lab group atStanford University (http://cmgm.stanford.edu/pbrown/) will beconstructed, and a scanner from Genetic Microsystems will be purchased.Arrays are hybridized with fluorophore-labeled cDNA as described below,scanned, and the data from a complete scan is reconstructed to yield apseudoimage (usually a TIFF image).

Production of DNA for arraying. The 20,000 member clonal ORF libraryobtained as described in Example VI will be used to prepare the DNA tobe arrayed. PCR products from various DNAs can be spotted onto slides.Alternatively, whole plasmid DNA purified from the 20,000 clones can beused directly for the production of microarrays, thus saving theconsiderable time and expense of the 20,000 PCR reactions. Eachmicroarray will contain a number of positive and negative controls.Positive controls will include genes known to be developmentallyregulated in T. cruzi, including amastin, porin and gp72 to representamastigote, trypomastigote and epimastigote expressed genes,respectively. T. cruzi genes for which we expect a relatively stablelevel of expression in the different life cycle stages include actin,tubulin, and GAPDH. The pORF.GFP vector without inserts and murine cDNAencoding cytokine genes will also be included as negative controls. Thecontrol DNAs to be included in the arrays will be produced by PCR usingsequence specific primers and genomic or plasmid DNA as template.

Spotting of DNAs in microarrays. Detailed protocols for most steps inthe process are readily available in the literature (J. DeRisi et al.,Nat. Genet. 14:457-460 (1996); J. DeRisi et al., Science 278:680-686(1997)). Slides are normally prepared using silanization or by coatingwith poly-L-lysine. The arrays will be probed with cDNAs isolated fromT. cruzi at different stages of development.

Sources of cDNA for screening. Synchronized cultures of parasites atdifferent time points during conversion from trypomastigotes toamastigotes will be obtained by acid induction of conversion of freshlyderived trypomastigotes or by harvesting Vero cells to which freshlyisolated trypomastigotes have been added for various periods of time.Initially, we will isolate mRNA from parasites or parasite-infected hostcells at one or two time points in the infection/conversion process,time 0 (immediately upon addition of trypomastigotes to host cells orjust prior to exposure to acid in the case of the acid-conversionprotocol), 12 hours and 24 hours post-infection/induction. Infectivetrypomastigotes not more than 2 hours post-release from heavily infectedVero cells cultured in T-75 flasks will be collected as “synchronized”trypomastigotes.

Production of labeled cDNAs. Brazil strain T. cruzi trypomastigotes frommonolayers of Vero cells at 37° C. in a 5% CO₂ atmosphere will beharvested by centrifugation of culture supernatants at 1,500×g for 10minutes at room temperature. Fresh T500 flasks containing semi-confluentmonolayers of Vero cells will be infected with 20 trypomastigotes percell (approximately 2.3×10⁸ parasites) and total RNA isolatedimmediately (to provide the baseline mRNA expression levels) or thecultures rinsed after 2 hours to remove non-infecting parasites and themRNA isolated at 12 or 24 hours post infection. Alternatively,amastigotes will be obtained by acid treatment of trypomastigotes:freshly harvested cell culture trypomastigotes are resuspended at5×10⁶/ml in DMEM containing 0.4% BSA (DMEM-BSA) buffered with 20 mM MES,pH 5 and incubated at 37° C. in 5% CO₂ for 0 (and harvested for baselinemRNA) or after 4 hours, centrifuged and resuspended inbicarbonate-buffered DMEM-BSA, pH 7.5 and incubated for another 8 or 16hours at 37° C. in 5% CO₂.

Total RNA is isolated by adding 5 mL of Ultraspec™ II RNA reagent(Biotecx Laboratories, Inc. Houston, Tex.) directly to each washed flaskand the resulting lysate is passed repeatedly through a pipette beforebeing transferred to a 10 ml polypropylene tube. The homogenate is heldon ice for 5 minutes to allow the dissociation of ribonucleoproteincomplexes. Total RNA is extracted by the addition of 1 ml chlorofolin.After vortexing for 15 sec, the sample is centrifuged at 12,000×g for 15minutes at 4° C. The aqueous phase (2.5-3.0 ml) is transferred to 1.5 mlpolypropylene microcentrifuge tubes, 0.5 vol of isopropanol is added,and the tubes are vortexed. To bind the RNA, 0.05 vol of RNATack™ resin(Biotecx Laboratories) is added and mixed by vortexing for 30 sec. Thetubes are spun for 1 minute in a microcentrifuge. The supernatant fluidis discarded and the pellet is washed twice with 75% ethanol. The pelletis dried briefly under vacuum to remove any traces of ethanol andresuspended in 0.1 vol of diethylpyrocarbonate (DEPC) treated water.After a brief spin to pellet the resin, the supernatant RNA istransferred to a fresh tube and quantitated by UV spectrophotometry.Approximately 300-400 ug of total RNA is obtained from one T500 flask.The method of total RNA isolation from axenically induced amastigoteforms is essentially the same except that the parasites are harvested bycentrifugation and the pellet is resuspended directly in Ultraspec™reagent at a density of 1×10⁸ cells per ml.

Labeled first strand cDNA is synthesized from the total RNA samplesusing Superscript™ II reverse transcriptase (BRL Life Technologies) andCyDye labeled fluorescent nucleotides (Cy3-dUTP and Cy5-dUTP, AmershamCorp.). In a sterile 0.5 ml microfuge tube first strand buffer, oligod(T)12-18, 10× low T dNTP mix, Cy3 or Cy5-dUTP (1 uM), DTT, Rnasin, andtotal RNA are added. The tube is incubated at 65° C. for 5 minutes andcooled to 42° C. Two microliters (400U) of Superscript™ II enzyme areadded and the reaction is held at 42° C. for 25 min. Another 2 ul ofenzyme are added, and the reaction is incubated for 35 minutes at 42° C.The reaction is stopped by the addition of 5 ul of 500 mM EDTA. Tohydrolyze residual RNA, 10 ul of 1 M NaOH are added and the sample isincubated at 65° C. for 1 hour. The sample is cooled to 25° C. and 25 ulof 1 M TrisHCl, pH 7.5 are added to neutralize the NaOH. The entirelabeled sample is transferred to a Microcon-30 microconcentrator(Amicon) and centrifuged at 14,000 rpm in a microfuge until the volumeis reduced to 20 ul.

Hybridization of arrays. Hybridization of the labeled cDNAs with themicroarrays is done in custom-made hybridization chambers (design andCAD drawings available from the Brown web page or completed units can bepurchased from TeleChem). The chambers are loaded with 5.0 μl ofhybridization buffer (to maintain conditions of 100% humidity in thecassette chamber thus preventing evaporation of the hybridizationsolution) prior to addition of the array slide. Approximately 10 μl ofhybridization solution containing the fluorophore-labeled cDNAs areadded to the array on the slide and a coverslip placed. Standardhybridization buffers for microarray experiments contain 5×SSC and 0.2%SDS. The hybridization chamber is then sealed and immersed in a hotwater bath (37° C.-70° C.) for 2-24 hours, depending on the specificapplication. Long hybridization times and low incubation temperaturesunder high salt conditions are used to favor the annealing of low copynumber sequences. Following the hybridization, the slides are removedfrom the hybridization cassette and rinsed in 0.2×SSC with and then in0.2×SSC without SDS, dried and scanned.

Data interpretation. To determine the genes that are upregulated duringthe conversion of trypomastigotes to amastigotes, we will cohybridize anarray with Cy3-labeled trypomastigote-derived cDNA and Cy5-labeledamastigote-derived cDNA. The relative green/red signal for each genewill then be determined for each spot on the array and the spots showinga relative increase in the Cy5 (red) over Cy3 (green) identified. Inthis case, we would expect the majority of spots in the array to exhibita 1:1 ratio of red:green, indicating a stable level of expression in thetwo life cycle stages. Spots showing a significant change from thisratio (>2:1) indicate upregulation in the amastigote stage relative tothe trypomastigote stage and will be selected for further study. Thislevel of sensitivity of change in expression level has been achieved inmultiple other studies using equipment and conditions similar to thosedescribed herein. To assure that the change in expression level observedin the initial analysis is real, the hybridization will be repeated withthe dyes “switched” on the two cDNA probes (i.e. Cy3-labeledamastigote-derived cDNA and Cy5-labeled trypomastigote cDNA). The changein expression on stage conversion will also be monitored by comparisonof the 12 hour and 24 hour amastigote samples.

Clone sequencing and gene identification. Clones identified as beingupregulated in the amastigote stage of the T. cruzi life cycle will beidentified, and those with the greatest relative increase in expressionselected as described above for further study. Up to 200 of the mosthighly upregulated clones will be end-sequenced (at the UGA MolecularGenetics Instrumentation Facility) for gene identification purposes.Clones containing ORFs for which no homologues can be identified basedon the clone-end sequencing will be fully sequenced. Using the completesequence information we will then conduct additional homology searchesin an attempt to establish the identity or putative function of the geneproducts. We will attempt a reasonably exhaustive electronic analysis ofeach protein sequence, following a standard set of protocols (e.g.,identification of repeats, signal sequences, GPI anchor addition sitesor transmembrane regions, domains, cluster analysis of homologues, etc.Based on the results of these determinations, a subset of these cloneswill be further investigated for protective function. Novel genes ofunknown function or with probable function which is consistent withservice as good vaccine candidates will be tested individually for theability to confer resistance to infection in a murine model of T. cruziinfection, as described in previous Examples. This work will befacilitated by the fact that the genes will already be in a vectorappropriate for DNA vaccination and thus no recloning of the insertswill be required.

Example VIII Production of a Targeted Polypeptide for Use in ProteinVaccine Against T. cruzi Infection and Disease

In this experiment, proteins are transduced at high efficiency intocells. For efficient transduction, the transducing substrates areprocessed such that they are capable of unfolding to traverse thebilipid membrane as a linear protein, in accordance with the method ofS. Schwarze et al. (Science 285:1569-1572 (1999); H. Nagahara et al.,Nature Med. 4:1449-1452 (1998)). For example, Schwarze et al. report thedelivery of biologically active TAT fusion protein to a mouse (Science285:1569-1572 (1999)). Thus, proteins capable of highly efficienttransduction are characterized as being unstable (high free energy, ΔG),misfolded and aqueous soluble. In many cases, shock folding from urea toaqueous on the ionic exchange column increases biological effect ofsome/most proteins 5-10 fold vs. dialysis. Dialysis, however, can beused as an alternative if desired or necessary.

The pTAT-produced proteins are typically completely insoluble (>99%) andare therefore stored in the inclusion bodies. This not only leads tohigh yields but safekeeping of the fusion proteins from the hostproteases. Shock-misfolding of the protein preferably takes place on theMono-Q/S and only requires the presence of TAT on the surface of theprotein, not on a correctly folded protein. TAT proteins are thought totransduce into cells in a linear array and then are likely refoldedand/or degraded by Hsp90 once inside the cell.

Buffers. Buffer Z=8 M urea/100 mM NaCl/20 mM HEPES (pH 8.0); Buffer A=50mM NaCl/20 mM HEPES (pH 8.0) or (pH 6.5); Buffer B=1 M NaCl/20 mM HEPES(8.0) or (pH 6.5).

Cloning into pTAT. cDNA encoding T. cruzi Lyt1 (porin; FIG. 19; SEQ IDNO:20) and TSA-1 was cloned into a polylinker of pTAT or pTAT-HA(obtained from S. F. Dowdy, Washington University) via restrictionenzymes or PCR. The TAT protein of HIV is known to cross cell membranes.The plasmid pTAT contains the TAT protein transduction domain and isused to produce urea-denatured genetic in-frame TAT fusion proteins (H.Nagahara et al., Nature Med. 4:1449-1452 (1998)). pTAT is an N′ terminalfusion so translation terminations in the 5′ UTR of cDNA were removed.The ATG in the NcoI site is in-frame.

pTAT Linker:

        NcoI      KpnI Agel        TCC ACC ATG GCC GGT ACC GGT CTC  (SEQ ID NO: 21) G  S   T   M   A   G   T   G   L    (SEQ ID NO: 22)   SphI     Eco   BstBI HindIII GAG GTG CAT GCG GTG AAT TCG AAG CTT  E   V   H   A   V   N   S   K   L

-   -   followed by 20 amino acids to TAA Ts termination codon.        pTAT-HA Linker:

The HA tag (underlined), flanked by glycine residues, was inserted intothe NcoI site of pTAT. The N′ NcoI site has been inactivated.

CC ATG TCC GGC TAT CCA TAT GAC GTC  (SEQ ID NO: 23)    M   S   G   Y   P   Y   D   V (SEQ ID NO: 24)CCA GAC TAT GCT GGC TCC ATG GCC . . .   P   D   Y   A   G   S   M   A

The ligated plasmid was transformed into a high copy number E. colistrain BL21. Supercoiled pTAT-cDNA “X” was transformed intoBL21(DE3)LysS strain (Novagen). Six to ten colonies were pulled for 1 mlovernight culture and IPTG was added. Bacteria were centrifuged at5000×g for 5 minutes at room temperature, the resuspended intoapproximately 300 ul 2×SDS sample buffer. The mixture was boiled andloaded onto two SDS-PAGE gels along with a negative control strain.After electrophoresis, one gel was stained with Coomassie Blue andtransferred, while the other was probed with anti-HA or cDNA-specificantibody. Although the pTAT leader is approximately 3.5 kD, proteinstypically migrate 5-10 kDa larger than their predicted fusion proteinsize on SDS-PAGE. High producing bacterial clones were identified, andglycerol stocks of the clones were made.

pTAT protein purification. 1 L of media was inoculated with 100-200 mlovernight culture of BL21 cells containing the pTAT-cDNA “X” plasmid ofinterest, and IPTG was added. The culture was rotated for about 4-6hours at 37° C. Cells were centrifuged at 5000×g for 5 minutes, the cellpellet was washed with ˜50 ml PBS(−) and centrifuged again. The pelletwas resuspended in 10 ml of buffer Z (8 M urea/100 mM NaCl/20 mM HEPES[pH 8.0]), then sonicated on ice 3×15 second pulses or until turbid. Themixture was clarified by centrifugation at 12,000×g for 10 minutes at 4°C. The supernatant brought to about 10-20 mM imidazole and added at roomtemperature to a pre-equilibrated 3-10 ml Ni-NTA (Qiagen) column inbuffer Z plus 10-20 mM imidazole. The flow was allowed to proceed bygravity or slight air pressure was applied via syringe as required. Theflow-through (FT) was collected. The column was then washed withapproximately 50 ml buffer Z plus 10-20 mM imidazole. The His-TAT-Xprotein was eluted by step-wise addition of 5-10 ml each 100, 250, 500,1 M imidazole steps in buffer Z. Fractions containing the protein wereidentified via SDS-PAGE or immunoblot using the 12CA5 antibody (anti-HA;Babco) and pooled. Proteins that did not bind at 10-20 mM imidazole,were passed through a second time at 5, 2 or 1 mM imidazole.

Ion exchange chromatography. Ion exchange chromatography was carried outon a 30 micron 10-40 ml ionic exchange column was packed with Resource Qresin (Pharmacia). The HIS-TAT-X protein was loaded onto the column bysyringe injection. The column was washed with approximately 40 ml bufferA, and the protein was eluted with a single step of 1 M NaCl. Fractionscontaining the protein were pooled and desalted on a PD-10 disposableG-25 Sephadex gravity column (Pharmacia) in HEPES (7.2) or PBS(+).

Basic proteins require a Mono S column compared with acidic proteinsoutlined above. The leader sequence confers a more basic nature to thefusion proteins. The amount of protein purified will dictate the columnsize required. Further, if the protein binds the Q resin at 8.0 but doesnot release with 1 M NaCl, the pH is reduced by 0.5 units until it stillbinds and is not present in the flow through and elutes with a highyield. In some cases proteins bind a Q resin at pH 6.5 and 7.0.Alternatively, one can start with an S resin at pH 6.5 and move up 0.5pH units as required.

Whether purified by dialysis or ionic exchange chromatography, aliquotsof ˜250 ul were flash frozen in 10-15% glycerol on dry ice/liquid. N₂and store at −80° C.

Cellular analyses. About ˜5-25 ug of protein was labeled with FITC(Molecular Probes cat. #F-1906) in 300 ul, 2 hour, room temperature inthe dark, following manufacturer's instructions. The labeled protein wasthen injected into a gel filtration column (S-12, S-6, S-200) in PBS ora PD-10 column (Pharmacia). Fractions containing purified labeledprotein were collected and pooled. About 100-400 ul of purified TAT-FITCfusion protein was added to ˜1×10⁶ cells in media/FBS (nonadherent cellsare best experimentally), and transduction was evaluated at t=0′, 15′,30′, 45′, 60′ on FACS (FL-1). The cells were analyzed directly in mediaor fix in 4% paraformaldehyde. The fixed cells are used for microscopy.Essentially 100% of the cells demonstrated intracellular localization ofthe protein.

The complete disclosures of all patents, patent applications includingprovisional patent applications, and publications, and electronicallyavailable material (e.g., GenBank amino acid and nucleotide sequencedeposits) cited herein are incorporated by reference. The foregoingdetailed description and examples have been provided for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed; many variations will be apparent to one skilled in the artand are intended to be included within the invention defined by theclaims.

1. A multicomponent vaccine comprising a plurality of polypeptides,wherein each polypeptide is a Trypanosoma polypeptide comprising aglycosylphosphatidylinositol anchor attachment site, or an immunogenicfragment of said Trypanosoma polypeptide; wherein administration of thevaccine is effective to treat or prevent Trypanosoma infection in amammal.
 2. The multicomponent vaccine of claim 1 wherein the Trypanosomais T. cruzi.
 3. The multicomponent vaccine of claim 1 wherein thepolypeptide is a member of the trans-sialidase family of proteins. 4.The multicomponent vaccine of claim 2 wherein the polypeptide isexpressed in a T. cruzi amastigote.
 5. The multicomponent vaccine ofclaim 4 wherein the polypeptide is selected from the group consisting ofTSA-1, ASP-1 and ASP-2.
 6. The multicomponent vaccine of claim 1comprising at least ten polypeptides.
 7. The multicomponent vaccine ofclaim 1 which stimulates at least one immune response in a mammalianhost selected from the group consisting of an antibody response and acell-mediated immune response.
 8. The multicomponent vaccine of claim 7which stimulates at least one of a Th1-biased CD4⁺ T cell response or aCD8⁺ T cell response.
 9. The multicomponent vaccine of claim 8 whichstimulates a CD8⁺ T cell response.
 10. The multicomponent vaccine ofclaim 7 which stimulates an antibody response, a Th1-biased CD4⁺ T cellresponse and a CD8⁺ T cell response.
 11. The multicomponent vaccine ofclaim 1 wherein the polypeptide comprises a membrane translocatingsequence.
 12. The multicomponent vaccine of claim 11 wherein themembrane translocating sequence is derived from HIV TAT protein.
 13. Themulticomponent vaccine of claim 1 further comprising a pharmaceuticallyacceptable carrier.